Tumor vaccine

ABSTRACT

The invention relates to the fields of medicine, immunology, and oncology. More specifically, the invention relates to methods and compositions for inducing an immune response against a tumor in an animal subject. The invention provides that a lung cancer cell or other tumor cells, genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen, and method for stimulating an immune response to a tumor with the tumor cell so genetically modified. The invention additionally provides a method of inhibiting a tumor, including a cancer such as lung cancer, by administering an allogeneic tumor cell, for example a cancer tumor cell such as a lung cancer tumor cell, genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 61/106,355, filed on Oct. 17, 2008.

FIELD OF THE INVENTION

The invention relates to the fields of medicine, immunology, and oncology. More specifically, the invention relates to methods and compositions for inducing an immune response against a tumor in an animal subject.

BACKGROUND OF THE INVENTION

Lung cancer is the most common cause of death due to cancer in the United States. For 2002, the American Cancer Society predicted that almost 170,000 new cases of lung cancer would be diagnosed and that 155,000 people would die from the disease. Patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) make up 70% of the newly diagnosed cases.

Current recommendations for patients with inoperable disease include platinum-based chemotherapy plus radiation therapy in locally advanced disease, or chemotherapy alone in patients with metastases. Typical response rates are between 15% to 30%, with median survivals of less than one year. Meta-analysis of 52 phase III clinical trials randomizing metastatic NSCLC patients between best supportive care and chemotherapy concluded that chemotherapy increases the chance of 1 year survival by 10% and the median survival by 6 weeks. A recent report from the Big Lung Trial group (BLT) reported similar results. The aggressiveness of NSCLC is thought to relate to its ability to evade the immune system perhaps by suppressing immune response priming by means of CD4 regulatory cells and/or by producing immunosuppressive cytokines such as TGF-β.

Thus, there exists the need to develop effective therapies to treat a tumor, including cancers such as lung cancer. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a tumor cell, for example, a lung cancer cell or other tumor cells, genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen. The invention also provides a method of stimulating an immune response to a tumor, including a cancer tumor such as a lung cancer tumor, by administering an allogeneic lung cancer tumor cell genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen. The invention additionally provides a method of inhibiting a tumor, including a cancer such as lung cancer, by administering an allogeneic tumor cell, for example a cancer tumor cell such as a lung cancer tumor cell, genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen. According to some embodiments of the invention, the vaccine is administered more than once.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B shows flow cytometry analysis. Panel A: Quality control of vaccine cells. Representative samples of vaccine cells coexpressing B7.1 (CD80) and HLA A1 (left panel) or HLA A2 (right panel) analyzed by flow cytometry. The percentage of double positive cells is indicated. CD80 and the HLA A allele must be coexpressed on 70% or more of the cells to qualify for immunization. Panel B. Patient CD8 cells purified for ELI-spot assays. Flow cytometry of a representative sample of patient CD8 (right panel) cells purified by negative selection and used for ELIspot analysis; the purity of cells is given in %. Left panel shows isotype control.

FIG. 2 shows analysis of CD8 immune response: Immunization of advanced lung tumor patients generates strong CD8 response. The frequency of IFN-y-spot forming CD8 cells obtained from lung tumor patients is plotted against the time on study in weeks. Immunizations were given every two weeks, zero representing the preimmunization status. 20,000 purified CD8 cells were used for ELI-spot assays. Panel A: Frequency of spot forming CD8 cells from HLA A1 and A2 positive patients challenged with HLA A1 or A2 transfected (matched) AD 100 tumor cells at a ratio of 20:1=CD8:AD100. Panel B: Frequency of spot forming CD8 cells from HLA A1 positive patients challenged with A2-AD100 or HLA A2-CD8 cells were challenged with A1-AD100 (mismatched). Panel C: Frequency of spot forming CD8 cells from non HLAA-1 or A2 patients cells challenged with A1 and A2 transfected AD100 (unmatched). Panel D: Frequency of spot forming CD8 cells from all patients challenged with untransfected wild type (w. t.) AD100 or, Panel E, with K562. Panel F: Mean frequency of spot forming CD8 cells from all patients challenged with any of the AD100 w. t. or transfected cells. Panel G: CD8 spot forming response of individual, clinically responding patients. The mean number of spots after restimulation with AD100 w. t., AD100-A1, AD100-A2, K562 or nothing in quadruplicate wells is plotted against time after study entry. Arrows indicate the time of last immunization. Patient 1004, 1007, 1010 contain follow up data analyzed at the points indicated after completion of nine immunizations (18 weeks). HLA type of each patient is indicated in brackets.

FIG. 3 shows the median survival time of all patients at the time of analysis. The median survival time was 18 months, exceeding the expected median survival time of less than one year for this group of patients.

FIG. 4 shows overall survival for the 19 B7 vaccine-treated non-small-cell lung cancer study patients.

FIGS. 5A and B show analysis of CD8 immune response. FIG. 5A (top two panels) shows CD8 prior to immunization or at 6,12 and 18 weeks after challenge with untransfected (AD wild type) vaccine cells or K562 control. FIG. 5B (lower six panels) shows CD8 response after termination of vaccination (arrow) in patients with clinical response.

FIG. 6 illustrates the sequence and annotation of one embodiment of a BPV-1-B7.1-HLA A1 vector.

FIG. 7 illustrates the survival curve from initiation of phase I clinical trial to present or to last survivor.

FIG. 8 illustrates patient response to different levels of vaccination. Patients who received a second or third course of vaccination fared better in terms of both clinical response and survival.

DETAILED DESCRIPTION

The invention relates to the discovery that administering allogeneic tumor cells expressing or caused to express CD80 (B7.1) and HLA antigens to cancer patients resulted in an anti-tumor immune response in the patients. More particularly, CD8-mediated immune responses were elicited in stage IIIB/IV NSCLC patients immunized several times with allogeneic NSCLC cells transfected with CD80 (B7.1) and HLA-A1 or A2. Immunization significantly increased the frequencies of interferon-Y-secreting CD8 T cells in all but one of the patients tested as discussed in more detail, below, in a clinical analysis of one set of patients, five of fourteen patients responded to immunization with stable disease or partial tumor regression. Further characterization was performed with additional patients.

Carcinoma of the lung is the leading cause of cancer death and the second most commonly occurring cancer in both men and women in the United States (Jemal, et al., CA Cancer J. Clin. 53: 5-43 (2003). Non-small-cell lung cancers (NSCLC) are considered to be minimally or nonimmunogenic, and may contain CD4 regulatory cells that suppress generation of cytotoxic lymphocytes (CTL) (Woo, et al., J. Immunol. 168: 4272-4276 (2002)). Although NSCLC has not been considered a good candidate for immunotherapy, the studies disclosed herein are based on the hypothesis that NSCLC is indeed suitable for successful vaccine therapy because the tumor cells have not been exposed to immune attack and have not yet developed resistance mechanisms.

Immunotherapy trials for lung cancer have previously yielded no consistent benefit in humans (Ratto, et al., Cancer 78: 244-251 (1996); Lissoni, et al., Tumori 80: 464-467 (1994); Ratto, et al., J. Immunother 23: 161-167 (2000)). Vaccine trials with B7.1 (CD80) transfected allogeneic or autologous cells have not been reported in patients with NSCLC prior to the studies disclosed herein, although similar vaccines have shown good activity in other human studies (Antonia, et al., J. Urol. 167: 1995-2000 (2002); Honig, et al., Cancer Immunol. Immunother. 49: 504-514 (2000); Hull, et al., Clin. Cancer. Res. 6: 4101-4109 (2000); von Mehren, et al., Clin. Cancer Res. 6: 2219-2228 (2000)). The objectives of the studies disclosed herein were to assess the safety, immunogenicity, and clinical response to an allogeneic whole cell tumor vaccine transfected with CD80 and HLA A1 or A2 administered to patients with advanced metastatic NSCLC. Disclosed herein are results on vaccine safety, clinical response, and overall survival.

As disclosed herein, to determine whether CD8 mediated immune responses could be elicited in stage IIIB/IV NSCLC patients, initially fourteen subjects were immunized several times with allogeneic NSCLC cells transfected with CD80 (B7.1) and HLA-A1 or A2. Additional patients were added. Patients enrolled were matched or unmatched at the HLA A1 or A2 locus and their immune response compared. Immunization significantly increased the frequencies of interferon-y secreting CD8 T cells in all but one patient in response to ex vivo challenge with NSCLC cells. The CDS response of matched and unmatched patients was not statistically different. NSCLC reactive CD8 cells did not react to IL562. Clinically, five of fourteen patients responded to immunization with stable disease or partial tumor regression. The study demonstrates that CD8IFN-y responses against non-immunogenic or immunosuppressive tumors can be evoked by cellular vaccines even at advanced stages of disease. The positive clinical outcome suggests that non immunogenic tumors may be highly susceptible to immune effector cells generated by immunization.

Thus, it has been discovered that the administration to a tumor patient of modified tumor cells expressing CD80 and an HLA antigen results in desirable therapeutic effects. Hence, in one embodiment, the invention provides a tumor lung cancer cell into which has been introduced a first nucleic acid encoding CD80 and a second nucleic acid encoding HLA antigen. These modified tumor cells can be administered more than once. The modified tumor cells can be administered 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times. Preferably, the vaccine is administered between 2 and 9 times.

As used in this specification, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. As used herein, unless specifically indicated otherwise, the word “or “is used in the “inclusive” sense of “and/or” and not the “exclusive” sense of” either/or.” In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise.

The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise (s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term “comprising” means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

The term “tumor” is used to denote neoplastic growth which may be benign (e.g., a tumor which does not form metastases and destroy adjacent normal tissue) or malignant/cancer (e.g., a tumor that invades surrounding tissues, and is usually capable of producing metastases, may recur after attempted removal, and is likely to cause death of the host unless adequately treated) (see Steadman's Medical Dictionary, 26th Ed, Williams & Wilkins, Baltimore, Md. (1995)).

The invention also provides a method of stabilizing or reversing a tumor load in a patient by administering to the patient an allogeneic tumor cell into which has been introduced a first nucleic acid encoding CD80 and a second nucleic acid encoding an HLA antigen.

In another embodiment, the invention provides a tumor cell, which can be a tumor cancer cell such as a lung cancer cell, genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen.

Exemplary HLA antigens include, but are not limited to, HLA A1, HLA A2, HLA A3, HLA A27, and the like. In a particular embodiment, the HLA antigen can be HLA A1 or HLA A2 (see Examples). One of skill in the art will appreciate that there are a number of different nucleic acid sequences encoding HLA antigens which may be used according to the invention without departing from the same (see below). Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention. However, preferred materials and methods are described. Materials, reagents and the like to which reference is made in the following description and examples are obtainable from commercial sources, unless otherwise noted.

Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined.

Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of recombinant DNA technology include for example, Ausubel et al., Current Protocols in Molecular Biology (Supplement 56), John Wiley & Sons, New York (2001); Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor (2001); Kaufman et al., Eds., Handbook of Molecular and Cellular Methods in Biology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). Standard reference works setting forth the general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Ed., McGraw Hill Companies Inc., New York (2001). The compositions according to the invention are optionally formulated in a pharmaceutically acceptable vehicle with any of the well known pharmaceutically acceptable carriers, including diluents and excipients (see Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, Mack Publishing Co., Easton, Pa. 1990 and Remington: The Science and Practice of Pharmacy, Lippincott, Williams & Wilkins, 1995). While the type of pharmaceutically acceptable carrier/vehicle employed in generating the compositions of the invention will vary depending upon the mode of administration of the composition to a mammal, generally pharmaceutically acceptable carriers are physiologically inert and non-toxic. Formulations of compositions according to the invention may contain more than one type of compound of the invention), as well any other pharmacologically active ingredient useful for the treatment of the symptom/condition being treated.

In some embodiments, the cancer cell can be a lung tissue cancer cell (also referred to as “lung cancer cell”) such as an adenocarcinoma cell type, for example, the lung cancer cell can be the AD 100 cell line, as exemplified hereinafter.

The invention additionally provides a method of stimulating an immune response to a tumor, for example, a cancer such as a lung cancer, in a patient by administering an allogeneic tumor cell genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen. The tumor cell can be a cancer cell, for example, a lung cancer tumor cell.

The methods of the present invention are intended for use with any subject that may experience the benefits of the methods of the invention. Thus, in accordance with the invention, “subjects”, “patients” as well as “individuals” (used interchangeably) include humans as well as non-human subjects, particularly domesticated animals.

In one embodiment, a method of the invention can include matching the HLA antigen to the individual administered the tumor lung cancer cell. Methods of determining HLA haplotypes are well known to those skilled in the art, for example, using well known serological assays using antibodies to HLA alleles or the mixed lymphocyte reaction. In a particular embodiment, a method of the invention can be performed with the HLA antigen HLA A1, HLA A2, HLA A3 or HLA A27. The methods of the invention cause various tumor cells (e.g., lung cancer cells) including, for example, an adenocarcinoma such as the AD100 cell line exemplified hereinafter.

In still another embodiment, the invention provides a method of inhibiting a tumor by administering an allogeneic tumor cell genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen. The tumor can be, for example, a cancer tumor cell such as a lung cancer tumor cell. In certain embodiments, the tumor inhibited is lung cancer by the administration of an allogeneic cancer cell modified to express CD80 (B7.1) and an HLA antigen.

As used herein, an “allogeneic cell” refers to a cell that is not derived from the individual to which the cell is to be administered, that is, has a different genetic constitution than the individual. An allogeneic cell is generally obtained from the same species as the individual to which the cell is to be administered. For example, the allogeneic cell can be a human cell, as disclosed herein, for administering to a human patient such as a cancer patient. As used herein, an “allogeneic tumor cell” refers to a tumor cell that is not derived from the individual to which the allogeneic cell is to be administered.

Generally, the allogeneic tumor cell expresses one or more tumor antigens that can stimulate an immune response against a tumor in an individual to which the cell is to be administered. As used herein, an “allogeneic cancer cell,” for example, a lung cancer cell, refers to a cancer cell that is not derived from the individual to which the allogeneic cell is to be administered. Generally, the allogeneic cancer cell expresses one or more tumor antigens that can stimulate an immune response against a cancer in an individual to which the cell is to be administered, for example, a lung cancer.

As used herein, a “genetically modified cell” refers to a cell that has been genetically modified to express an exogenous nucleic acid, for example, by transfection or transduction. A cell can be genetically modified to express, for example, a nucleic acid encoding CD80 (B7.1) and/or a nucleic acid encoding an HLA antigen, as disclosed herein. When a cell is to be genetically modified to express more than one polypeptide, for example, CD80 (B7.1) and an HLA antigen, it is understood that the polypeptides can be encoded on separate nucleic acids (see Example 1) or on the same nucleic acid, if desired. Methods of genetically modifying a cell are well known to those skilled in the art.

The invention provides methods and compositions for stimulating an immune response in a cancer patient. The compositions and methods are particularly useful for stimulating an immune response against non-immunogenic tumors. As used herein, a non-immunogenic tumor is a tumor that does not elicit a spontaneous immune response detectable, for example, by appreciable stimulation of CD8 T cells that produce interferon-γ (IFNγ) in tumor infiltrating lymphocytes (TILs).

Traditionally, melanoma and other immunogenic tumors have been preferred for treatment by immunotherapy. In the present invention, non-immunogenic tumors are considered good targets for active immunotherapy because the tumor cells have not been immuno-selected for evasion of the CTL response. Exemplary non-immunogenic tumors include, but are not limited to, lung, pancreatic, and the like.

A particularly useful nonimmunogenic tumor type is non small cell lung cancer (NSCLC), as exemplified herein. NSCLC tumors are good targets for active immunotherapy because they are non-immunogenic and do not spontaneously generate CTL responses. Therefore, NSCLC tumor cells have not developed evasive mechanisms towards cytotoxic T and natural killer (NK) cells, and NSCLC tumors are susceptible to cytotoxic attack. As disclosed herein, a composition of the invention was used to successfully slow tumor growth in NSCLC patients (see Examples II and III).

NSCLC tumors can also be genetically engineered to express and secrete gp96 and enhance the effectiveness of a vaccine because it combines adjuvant activity with polyvalent peptide specificity. Polyvalence prevents immunoselection and evasion. Tumor secreted gp96 activates dendritic cells (DC), natural killer cells (NK) and cytotoxic T lymphocytes (CTL), activating innate and adaptive immunity. Tumor cells can be killed by NK-specific mechanisms, by promiscuous killing of CD8 CTL through NKG2D, and by MHC restricted CD8 CTL activity. The activation of DC and NK by tumor secreted gp96 may also counteract the generation of immuno-suppressive CD4 regulatory cells found in NSCLC tumors. Tumor secreted gp96 stimulates, antigen cross presentation via the CD91 receptor on DC and macrophages. NSCLC are known to share tumor antigens also found in melanoma and may be endowed with additional shared antigens. Therefore allogeneic, gp96 secreting tumor cells used as vaccine are expected to generate immunity to the patient's autologous tumor. Similarly, a composition of the invention containing an allogeneic tumor cell expressing CD80 and an HLA antigen can generate immunity to the patient's autologous tumor.

Lung tumors prevent priming of CTL by regulatory cells, by TGF-P secretion and by down regulation of MHC class I. Therefore, immunogenic vaccines are needed to generate a CTL response. Lung tumors are susceptible to CTL killing because they have not been selected for CTL evasion. Lung tumor TIL contain large numbers of CD4 regulatory cells suppressing priming. In contrast, melanoma TIL contain antigen specific CD8 CTL whose killing activity has been blocked, indicating that priming has taken place already. As disclosed herein, lung cancer patients were successfully treated with a vaccine containing an allogeneic tumor cell genetically modified to express CD80 (B7.1) and an HLA antigen (Examples II and III). Thus, immunotherapy (vaccine therapy) of NSCLC is useful for treating this otherwise deadly disease.

As disclosed herein, an adenocarcinoma is an exemplary lung cancer that can be used in compositions and methods of the invention to express CD80 (B7.1) and an HLA antigen. Other types of lung cancer are well known, and cells derived from other types of lung cancers can be similarly used in compositions and methods of the invention. Exemplary lung cancers include, for example, non-small cell lung cancer, which can be adenocarcinoma, squamous cell carcinoma, or large cell carcinoma, small cell lung cancer, and carcinoids. One skilled in the art can readily obtain tissue samples from various types of lung cancers and generate a cell line useful for treating a lung cancer, using methods similar to those disclosed herein. Similarly, other types of nonimmunogenic tumors can be used to generate allogeneic tumor cells that can be genetically modified to express CD80 (B7.1) and an HLA antigen and used to treat a similar type of tumor or a tumor expressing similar types of tumor antigens.

An exemplary allogeneic tumor cell is the AD 100 cell line, which is a human lung adenocarcinoma cell line, as disclosed herein. Other lung cancer cell lines are well known to those skilled in the art and can be similarly used to generate an allogeneic cell genetically modified with CD80 (B7.1) and an HLA antigen. For example, numerous cell lines, including lung cancer cell lines are well known and available from the American Type Culture Collection (ATCC; Manassas Va.). Exemplary NSCLC cell lines include, but are not limited to, NCI-H2126[H2126] (ATCC CCL-256); NCI-H23 [H23] (ATCC CRL-5800); NCI-H1299[H1299] (ATCC CRL-5803); NCI-H358 [H358] (ATCC CRL-5807); NCI-H810 [H810] (ATCC CRL-5816); NCI-H522 [H522] (ATCC CRL-5810); NCI-H1155 [H1155] (ATCC CRL-5818); NCI-H647 [H647] (ATCC CRL-5834); NCI-H650 [H650] (ATCC CRL-5835); NCI-H838[H838] (ATCC CRL-5844); NCI-H920 [H920] (ATCC CRL-5850); NCI-H969 [H969] (ATCC CRL-5852); NCI-H1385 [H1385] (ATCC CRL-5867); NCI-H1435[H1435] (ATCC CRL-5870); NCI-H1437[H1437] (ATCC CRL-5872); NCI-H1563[H1563] (ATCC CRL-5875); NCI-H1568[H1568] (ATCC CRL-5876); NCI-H1581[H1581] (ATCC CRL-5878); NCI-H1623[H1623] (ATCC CRL-5881); NCI-H1651 [H1651] (ATCC CRL-5884); NCI-H1693[H1693] (ATCC CRL-5887); NCI-H1703[H1703] (ATCC CRL-5889); NCI-H1734[H1734] (ATCC CRL-5891); NCI-H1755[H1755] (ATCC CRL-5892); NCI-H1770 [H1770] (ATCC CRL-5893); NCI-H1793[H1793] (ATCC CRL-5896); NCI-H1838[H1838] (ATCC CRL-5899); NCI-H1869[H1869] (ATCC CRL-5900); NCI-H1915 [H1915] (ATCC CRL-5904); NCI-H1944[H1944] (ATCC CRL-5907); NCI-H1975[H1975] (ATCC CRL-5908); NCI-H1993 [H1993] (ATCC CRL-5909); NCI-H2023[H2023] (ATCC CRL-5912); NCI-H2030 [H2030] (ATCC CRL-5914); NCI-H2073 [H2073] (ATCC CRL-5918); NCI-H2085 [H2085] (ATCC CRL-5921); NCI-H2087 [H2087] (ATCC CRL-5922); NCI-H2106 [H2106] (ATCC CRL-5923); NCI-H2110 [H2110] (ATCC CRL-5924); NCI-H2135 [H2135] (ATCC CRL-5926); NCI-H2172[H2172] (ATCC CRL-5930); NCI-H2228 [H2228] (ATCC CRL-5935); NCI-H2291 [H2291] (ATCC CRL-5939); NCI-H2342 [H2342] (ATCC CRL-5941); NCI-H2347 [H2347] (ATCC CRL-5942); NCI-H2405 [H2405] (ATCC CRL-5944); NCI-H2444 [H2444] (ATCC CRL-5945); and NCI-H2122 [H2122] (ATCC CRL-5985). These and other tumor cell lines, particularly those of nonimmunogenic tumors, can similarly be used in compositions and methods of the invention.

As disclosed herein, these and other tumor cell lines can be genetically modified to express exogenous molecules that enhance an immune response to tumor antigens. Such molecules include, but are not limited to, CD80 (B7.1), human HLA antigens, for example, HLA A1, A2, A3, A27, and the like. One skilled in the art can readily obtain appropriate sequences encoding such molecules using well known methods. One skilled in the art will readily understand that variants of such molecules are available or can be readily obtained using well known methods. Based on known complete or partial sequences, one skilled in the art can use well known molecular biology methods to obtain nucleic acid sequences suitable to genetically modify a tumor cell, as disclosed herein. It is understood that these exemplary sequences as well as natural variations of such sequences are considered within the scope of the invention.

Exemplary nucleic acid sequences encoding molecules that enhance an immune response are available, for example, from GenBank, including complete and partial cDNA sequences as well as genomic sequences, and such sequences can be used to obtain nucleic suitable nucleic acid sequences encoding desired immune enhancing molecules. A representative selection of such sequences available from GenBank include, but are not limited to, GenBank accession numbers NT_(—)005612; NM_(—)012092; NM_(—)175862; NM_(—)006889; NM_(—)005191; BC_(—)042665; NM_(—)012092; NM_(—)175862; NM_(—)006889; NM_(—)152854; NM_(—)005214; NM_(—)005514; NM_(—)002116; Z70315; NM_(—)002127; AH013634; L34703; L34734; AF389378; U30904; AH006709; AH006661; AH006660; X55710; U04244; U35431; M24043; U03859; NM_(—)005514; NM_(—)002116; Z30341; NM_(—)012292; NM_(—)002127; NM_(—)002117; AH007560; AH000042; AB048347; AB032594; AJ293264; AJ293263; AB030575 AB030574; AB030573; AF221125; AF221124; AH009136; X60764; AB032597; L17005; Y13267; AH003586; Z46633; Z27120; Z33453; Z23071; X02457; X57954; K02883; U21053; U04243; U18930; L36318; L36591; L38504; L33922; M20179; M20139; M24042; M15497; M31944; U04787; U01848; M27537; U11267; U03907; U03863; U03862; U03861; NM002116; L34724; L34723; L34721; L34737; L34701; Z97370; L15370; AH003070; M20179; M16273; M16272; M15497; M19756; M19757; NT008413, and the like.

The compositions and methods of the invention are useful for stimulating an immune response against a tumor. Such immune response is useful in treating or alleviating a sign or symptom associated with the tumor. Such an immune response can ameliorate a sign or symptom associated with a lung cancer. As used herein, by “treating” is meant reducing, preventing, and/or reversing the symptoms in the individual to which a compound of the invention has been administered, as compared to the symptoms of an individual not being treated according to the invention. A practitioner will appreciate that the compositions and methods described herein are to be used in concomitance with continuous clinical evaluations by a skilled practitioner (physician or veterinarian) to determine subsequent therapy. Hence, following treatment the practitioners will evaluate any improvement in the treatment of the pulmonary inflammation according to standard methodologies. Such evaluation will aid and inform in evaluating whether to increase, reduce or continue a particular treatment dose, mode of administration, etc.

The methods of the invention can thus be used to treat a tumor, including, for example, a cancer such as a lung cancer. The methods of the invention can be used, for example, to inhibit the growth of a tumor by preventing further tumor growth, by slowing tumor growth, or by causing tumor regression. Thus, the methods of the invention can be used, for example, to treat a cancer such as a lung cancer. It will be understood that the subject to which a compound of the invention is administered need not suffer from a specific traumatic state. Indeed, the compounds of the invention may be administered prophylactically, prior to any development of symptoms (e.g., a patient in remission from cancer). The term “therapeutic,” “therapeutically,” and permutations of these terms are used to encompass therapeutic, palliative as well as prophylactic uses. Hence, as used herein, by “treating or alleviating the symptoms” is meant reducing, preventing, and/or reversing the symptoms of the individual to which a therapeutically effective amount of a composition of the invention has been administered, as compared to the symptoms of an individual receiving no such administration.

The term “therapeutically effective amount” is used to denote treatments at dosages effective to achieve the therapeutic result sought. Furthermore, one of skill will appreciate that the therapeutically effective amount of the composition of the invention may be lowered or increased by fine tuning and/or by administering more than one composition of the invention (e.g., by the concomitant administration of two different genetically modified tumor cells), or by administering a composition of the invention with another compound to enhance the therapeutic effect (e.g., synergistically). The invention therefore provides a method to tailor the administration/treatment to the particular exigencies specific to a given mammal. As illustrated in the following examples, therapeutically effective amounts may be easily determined for example empirically by starting at relatively low amounts and by step-wise increments with concurrent evaluation of beneficial effect. The methods of the invention can thus be used, alone or in combination with other well known tumor therapies, to treat a patient having a tumor. One skilled in the art will readily understand advantageous uses of the invention, for example, in prolonging the life expectancy of a lung cancer patient and/or improving the quality of life of a lung cancer patient.

Current recommendations for NSCLC patients with locally-advanced inoperable disease (stage IIIB) include platinum-based chemotherapy plus radiation therapy, and chemotherapy alone for patients with metastases (stage IV) (Clinical practice guidelines for the treatment of unresectable non-small-cell lung cancer; adopted on May 16, 1997 by the American Society of Clinical Oncology, J. Clin. Oncol. 15: 2996-3018, 1997). Results of these approaches are nevertheless poor, and the increase in survival is limited. The largest meta-analysis published to date concluded that chemotherapy increases the chance of 1-year survival by 10% and median survival by 6 weeks (Chemotherapy in non-small cell lung cancer: A meta-analysis using updated data on individual patients from 52 randomizsed clinical trials. Non-Small Cell Lung Cancer Collaborative Group. BMJ 311: 899, 1995). A recent report from the Big Lung Trial group (BLT) reported similar results (Stephens et al., Proc. Am. Soc. Clin. Oncol. 21: 2002 (abstract 1661)). In phase III clinical trials, patients with metastatic disease have a median survival of less than 1 year (Schiller, et al., N. Engl. J. Med. 346: 92-98 (2002)).

Two phase III trials showed that after failure of first-line chemotherapy, only 6% of patients receiving standard second-line chemotherapy could expect to respond, with median survival being approximately 6 months (Shepherd, et al., J. Clin. Oncol. 18: 2095-2103 (2000); Fossella, et al., J. Clin. Oncol. 18: 2354-2362 (2000)). In the experiments described herein, the group of patients had a very poor prognosis as a result of their relapsed or metastatic disease status, and most patients had been unsuccessfully treated with surgery, radiation, and/or palliative chemotherapy, resulting in a projected survival of less than 6 months.

A vaccination approach such as that disclosed herein can be an effective means of inducing immune response in patients with nonimmunogenic tumors. There is evidence that NSCLC tumors contain tumor antigens (Yamazaki, et al., Cancer Res. 59: 4642-4650 (1999); Weynants, et al., Am. J. Respir. Crit. Care Med. 159: 55-62 (1999); Bixby, et al., Int. J. Cancer 78: 685-694 (1998); Yamada, et al., Cancer Res. 63: 2829-2835 (2003)). However, it has been thought that lung tumors are poor candidates for immunotherapy because they are poorly immunogenic and are potentially immunosuppressive (Woo, et al., J. Immunol. 168: 4272-4276 (2002); Woo et al., Cancer Res. 61: 4766-4772 (2001); Neuner, et al., Int. J. Cancer. 101: 287-292 (2002); Neuner, et al., Lung Cancer 34 (supplement 2): S79-82 (2001); Dohadwala, et al., J. Biel Chem. 276: 20809-20812 (2001)), thereby anergizing or tolerizing T-cells (Schwartz, J. Exp. Med. 184: 1-8 (1996); Lombardi, et al., Science 264: 1587-1589 (1994)). Lung tumors, therefore, have not been subjected to immune attack, and hence have not been able to evolve evasive mechanisms to resist immune effector cells. Lung tumors, unlike immunogenic tumors that harbor tumor-infiltrating lymphocytes, thus may succumb to killer CTLs, especially in light of the involvement of CD8 CTLs in tumor rejection in a number of model systems (Podack, J. Leukoc. Biel. 57: 548-552 (1995)).

As disclosed herein, an allogeneic whole cell vaccine was chosen because whole cell. Vaccines have given the best clinical results so far. For example, statistically significant survival benefit occurred when a whole cell melanoma vaccine was administered (Morton, et al., Ann Surg. 236: 438-449 (2002)). In contrast, vaccine directed at a single epitope may have limited utility due to tumor escape mutants (Velders, et al., Semin Oncol. 25: 697-706 (1998)). The additional advantage of a whole cell vaccine approach is that it does not require a priori delineation of specific lung tumor antigens. If vaccination is successful and CTLs are generated, as was found in the experiments disclosed herein, the responsible antigenic sites can be identified later. Allogeneic cell-based vaccines offer a good alternative to autologous vaccines under the assumption that lung tumor antigens are shared in lung tumors of different patients, and the antigens can be cross-presented by the patients' antigen-presenting cells. Although there is only limited evidence for shared antigens in lung tumors (Yamazaki, et al., Cancer Res. 59: 4642-4650 (1999); Yamada, et al., Cancer Res. 63: 2829-2835 (2003)), this has been shown in other tumors (Fong, et al., Annu. Rev. Immunol. 18: 245-273 (2000); Boon, et al., Annu. Rev. Immunol. 12:337-365 (1994)).

To obtain direct evidence that the CD8 cells generated in response to allogeneic vaccination recognize autologous tumor cells, tumor specimens should be obtained at the time of surgery. Tumor specimens were not available in the trial of patients disclosed herein with advanced disease (see Examples I1 and 111). However, the prolonged maintenance of a high frequency of patient CD8 cells reacting to AD100 in vitro, and their increase in some patients (No. 1004 and No. 1007; FIG. 5) even after cessation of external vaccination, is consistent with the immune stimulation of patient CD8 cells by the autologous tumor and their cross-reaction with the allogeneic vaccine.

In the experiments disclosed herein, although only one patient had a partial response, five other patients had stable disease. Enhanced immune reactivity was demonstrated by a CD8-mediated tumor-specific immune response. The fact that six (32%) of 19 patients with very poor prognosis exhibited disease stabilization of a rapidly lethal condition, with median survival of the whole cohort reaching 18 months despite far-advanced disease, is encouraging. The results disclosed herein indicate that tumor progression is slowed by vaccination, and that this effect occurs regardless of whether or not patients are allogeneic to the HLA A1 or A2 locus of the vaccine. The findings also indicate that indirect antigen presentation can be effective in promoting antitumor activity and that allogeneic MHC molecules enhance the effect.

In the results disclosed herein, the vaccine was well tolerated and the patients' quality of life was very good, thus improving patient outcome. Because this is an immunologic product, it was assumed that some immune-mediated side effects would be anticipated. Probable examples of such phenomena of expected tolerable side effects were, for example, the local erythema at the vaccination site in five patients, and the episode of arthritic pain experienced by one patient (see Example 3).

A composition of the invention containing a tumor cell genetically modified to express CD80 and an HLA antigen can be combined with a physiologically acceptable carrier useful in a vaccine by including any of the well known components useful for immunization. The components of the physiological carrier are intended to facilitate or enhance an immune response to an antigen administered in a vaccine. The formulations can contain buffers to maintain a preferred pH range, salts or other components that present the antigen to an individual in a composition that stimulates an immune response to the antigen. The physiologically acceptable carrier can also contain one or more adjuvants that enhance the immune response to the antigen. Formulations can be administered subcutaneously, intramuscularly, intradermally, or in any manner acceptable for immunization.

An adjuvant refers to a substance which, when added to an immunogenic agent of the invention such as tumor cell genetically modified to express CD80 and an HLA antigen, nonspecifically enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture. Adjuvants can include, for example, oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts), liposomes and microparticles, such as, polysytrene, starch, polyphosphazene and polylactide/polyglycosides.

Adjuvants can also include, for example, squalene mixtures (SAF-I), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. Nature 344: 873-875 (1990). For veterinary use and for production of antibodies in animals, mitogenic components of Freund's adjuvant (both complete and incomplete) can be used. In humans, Incomplete Freund's Adjuvant (IFA) is a useful adjuvant. Various appropriate adjuvants are well known in the art (see, for example, Warren and Chedid, CRC Critical Reviews in Immunology 8: 83 (1988); Allison and Byars, in Vaccines: New Approaches to Immunological Problems, Ellis, ed., Butterworth-Heinemann, Boston (1992)). Additional adjuvants include, for example, bacille Calmett-Guerin (BCG), DETOX (containing cell wall skeleton of Mycobacterium phlei (CWS) and monophosphoryl lipid A from Salmonella minnesota (MPL)), and the like (see, for example, Hoover et al., J. Clin. Oncol., 11: 390 (1993); Woodlock et al., J. Immunotherapy 22: 251-259 (1999)).

FIG. 6 illustrates the sequence and annotation of one embodiment of a BPV-1-B7.1-HLA A1 vector derived from a bovine papillomavirus type 1 (BPV-1) vector. The vector was further engineered to contain two expression cassettes for expression genes under the CMV and the Metallothioneine promoter, respectively. The sequence of this vector is shown at the end of the specification.

The compositions and methods of the invention disclosed herein are useful for treating a patient having a tumor. Although particular embodiments are exemplified with lung cancers, it is understood that a similar approach can also be used to treat other types of tumors, including cancers, using suitable allogeneic cells.

It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention. While the claimed invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made to the claimed invention without departing from the spirit and scope thereof. Thus, for example, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Example 1 Allogeneic Vaccination with a B7.1/HLA-A Gene-Modified Adenocarcinoma Cell Line in Patients with Advanced Non-Small-Cell Lung Cancer

This example describes the protocol used for allogeneic vaccination with a B7.1 HLA-A gene-modified adenocarcinoma cell line in patients with advanced non-small-cell lung cancer (NSCLC). This example describes the experimental protocol used.

The following experiments were designed (a) to measure whether CD80 and HLA A transfected, allogeneic lung tumor cells used for immunotherapy can elicit tumor specific CD8-CTL activation and expansion, assessed by ELIspot for IFN-γ; (b) to evaluate the safety and toxicity of administering allogeneic tumor cell vaccines transfected with B7.1 and HLA A1 or A2 in patients with Non-Small Cell Lung Carcinoma (NSCLC); and (c) to evaluate the antitumor effect of this B7.1 vaccine in clinical outcomes for patients with NSCLC.

Selection of Patients. Initially, fifteen patients with newly diagnosed or relapsed metastatic non-small cell lung cancer (NSCLC) were treated. The analysis of these 15 patients is described in Example 2. An additional four patients were added, for a total of 19 patients, and the further results with the 19 patients are described in Example 3. The patients had already failed chemotherapy, radiotherapy, surgery or a combination of all. Eligibility criteria were as follows: age>18 years, Eastern Cooperative Oncology Group (ECOG) performance status 0-2, measurable disease, signed informed consent, and histologically confirmed NSCLC (stage IIIB with malignant pleural effusion, stage IV, or recurrent). Patients with brain metastasis were included if these were already treated. Patients were not eligible for study if they were receiving chemotherapy, radiation therapy or a biologic modifying agent or during the preceding 4 weeks. All patients were treated in the outpatient clinic at Sylvester Comprehensive Cancer Center/University of Miami. A complete history and physical exam was performed, including weight and vital signs, with performance status assessed by ECOG criteria. The following tests were performed prior to enrollment: complete blood count; platelet count; chemistries (uric acid, calcium, phosphorus, transaminases including serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT), alkaline phosphatase, lactate dehydrogenase (LDH), total and direct bilirubin, blood urea nitrogen (BUN), creatinine, albumin, total protein, electrolytes, and glucose); and electrocardiogram (EKG). HLA typing was obtained. Patients were followed twice monthly while being vaccinated, with tumor response assessed by computed tomography (CT) scans. Tumor measurements were obtained from the results of radiographic studies, including CT scans of relevant sites.

Vaccine Cell Line and Genetic Modification. A human lung adenocarcinoma cell line was established in 1994 by Dr. N. Savaraj (University of Miami, Department of Medicine) from a biopsy of a lung cancer patient, designated as AD100. The patient was a 74 year old white male who presented in 1993 with initial symptoms of pelvic pain from bone erosion of the iliac crest due to metastatic pulmonary adenocarcinoma. Cancer cells for culture were obtained by bone marrow aspiration from the area of pelvic bone destruction. The patient was treated with radiation therapy to the pelvis, but expired one month after diagnosis. The cell line derived from this patient has been kept in culture in standard medium (described below) and is free of contamination by mycoplasma, virus or other adventitious agents. The cell line is homogeneous, adherent to plastic, and grows with a rate of division of approximately 26 h.

Genetic Modification. AD100 was transfected with plasmid cDNA, pBMG-Neo-B7.1 and pBMG-His-HLA A2 or with B45-Neo-CM-A1-B7.1 (Yamazaki et al., Cancer Res., 59: 4642, 1999) Transfected cells were selected with G418 and histidinol. Verification of correct sequences was based on restriction analysis and the expression of the relevant gene products, namely G418 or histidinol resistance for the vector sequence, HLA A1, A2, and B7.1 expression for the transfected cDNA. The cells were irradiated to prevent their replication, for example, with 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in 10% DMSO in aliquots of 5×107 cells until use. Upon replating in tissue culture the cells appeared viable for about 14 days but were unable to form colonies, indicating their inability to replicate. They were therefore considered safe for use as vaccine cells. The minimum requirement for their use as vaccine was the co expression of HLA A1 or A2 plus B7.1 on at least 70% of the cells, as shown in FIG. 1A for representative batches of vaccine cells. The untransfected AD100 line was negative by FACS for staining with anti HLA A1 or A2 or B7.1. FIG. 1A shows the quality control by flow cytometric analysis of CD80 and HLA A1 or A2 transfected AD100 vaccine cells used for immunization.

Immunizations. Intracutaneous injections were given at multiple body sites to reduce the extent of local skin reactions. Patients who were HLA A1 or A2 received the corresponding HLA-matched vaccine, whereas patients who were neither HLA A1 nor HLA A2 received HLA A1-transfected vaccine (that is, HLA-unmatched vaccine). On a given vaccination day, the patient received the total dose of 5×107 irradiated cells (12,000 rad) divided into two to five aliquots for administration as two to five intradermal injections of each aliquot in an extremity, spaced at least 5 cm at needle entry from the nearest neighboring injection. A total of nine immunizations (4.5×108 cells) were given over the course of therapy, one every two weeks, provided that no tumor progression occurred under therapy (Table 1). On subsequent vaccinations, the injection sites were rotated to different limbs in a clockwise manner. One course of vaccination comprised three biweekly injections. Patients with evidence of stable disease or responding NSCLC by imaging evaluation (CT Scans) and none to moderate toxicity (grade<2) were treated with an additional course at the same dose. The second course of injections started two weeks after the third vaccination that completed the first course. In the absence of tumor progression by CT scans and with no severe or life-threatening toxicity (grade>3), a third course at the same dose of therapy was given, starting two weeks after the third vaccination of the second course of therapy. Clinical” toxicity, and immunologic evaluations by blood tests prior to and after each course were performed was done. Patients were followed clinically weekly during the study, including monitoring blood counts and basic chemistries (Table 1).

Table 1 shows the treatment and evaluation schedule of NSCLC (IIIB/IV) patients. Patients were immunized nine times in biweekly intervals, as discussed above. Immunological assays were done prior to and after each of three immunizations.

TABLE 1 Immunizations and Immunological Evaluations Study Entry Course 1 Course 2 Course 3 Weeks on Study 1 2 4 6 7 8 10 12 13 14 16 18 19 Pre-Entry Evaluation x Immunization # 1 2 3 4 5 6 7 8 9 Clinical Evaluation x x x x x x x x x x x x x Toxicity Evaluation x x x x x x x x x x x x Immunological Evaluation # 1 2 3 4

Immunological Testing Immunological tests were performed included skin tests delayed-type hypersenstivity (DTH) and enzyme-linked immunospot (ELISPOT) assays for interferon-γ IFN-γ. Immune responses mediated by CD4 cells were examined by DTH-reaction following intradermal injection of 105A1, A2 or untransfected AD100-B7 vaccine cells. Purified CD8 cells were obtained from patients prior to and after each course of three immunizations. CDS cells. Were enriched by negative depletion with anti-CD56, anti-CD4 and other antibodies using the Spin-sep prep (Stem Cell Technologies; Vancouver, Canada). Purity was better than 80% (FIG. 1B) the primary contaminating cells being B cells (not shown). CD8 cells were frozen in 10% dimethylsulfoxide (DMSO) and 20% fetal calf serum (FCS) containing medium for analysis until all vaccinations of a study patient were completed. Analysis for pre-immune and post-vaccination ELISPOT frequency was carried out on the same day in the same micro titer plate. Assays were done in quadruplicate, stimulating 2×104 purified patient CD8 cells with, respectively, 103 A1 or A2 transfected or untransfected AD100, with K562 or with media only for three days and determining the frequency of IFN-y producing cells by ELISPOT. Immune assays were performed prior to immunization and after 3, 6, and 9 immunizations.

Statistical Analysis. Patient characteristics are presented as counts with percentages, or as mean values and range. Overall survival, estimated by the Kaplan-Meier product-limit method, is defined as time from enrollment onto study until death from any cause. In the absence of death, follow-up was censored at the date of last patient contact. Univariate and multivariate proportional hazards regression were used to determine whether patients' survival time was related to age (continuous), sex, race (other versus white non-Hispanic), tumor pathology (adenocarcinoma versus other), and HLA-matching of vaccine. Logistic regression was used for the corresponding analyses of clinical response. For hazard ratios and the percentage of patients surviving, 90% confidence intervals (CIs) L₉₀-U₉₀ are reported. These can be interpreted as providing 95% confidence that the parameter being estimated, such as the hazard ratio, exceeds L₉₀.

Example 2 Specific CD8 T Cell Response of Advanced Lung Cancer Patients to Whole Cell Immunization with an Allogeneic Vaccine

This example describes the results of a 15 patient group study on whole cell immunization with an allogeneic vaccine.

Patients with advanced NSCLC stage IIIB/IV were HLA typed. HLA A1 positive patients received the AD-A1-B7 vaccine; HLA A2 positive patients received the AD-A2-B7 vaccine; and patients that were neither HLA A1 nor A2 positive received either the AD-A1-B7 or AD-A2-B7 vaccine. The frequency of IFN-y secreting CD8 cells was determined by ELISPOT after restimulation of purified patient-CD8 cells in vitro with HLA A1 or A2 transfected or untransfected AD100. Controls included stimulation with K562 and incubation of CD8 cells without stimulator cells.

ELISPOT responses of immunized tumor patients are presented as HLA matched responses (FIG. 2A), representing the number of IFN-γ secreting CD8 cells obtained from HLA A1 or A2 patients challenged in vitro for three days with HLA A1 or A2 transfected AD100 cells, respectively. HLA mismatched responses indicate the number of spots formed when CD8 cells from A1 or A2 patients were challenged with A2 or A1 transfected AD100, respectively (FIG. 2B). The matched response increased 15-fold, from 6±4 (standard error of the mean, SEM) IFN-γ secreting, pre-immune CD8 cells (per 20 thousand) to maximal 90±35 (SEM) IFN-γ secreting cells after six immunizations and remained at this level during the next three immunizations. The mismatched response increased 5.7 fold, from 24±18 to 142±42 maximal. Included in this group of nine patients is the one patient who showed no response (0 spots) before or after three immunizations, at which time the tumor progressed and the patient was taken off trial.

The remaining 5 patients were negative for HLA A1 or A2. These patients CD8 response to challenge with A1 or A2 transfected AD 100 is shown as unmatched response in FIG. 2C. The frequency of IFN-γ secreting CDS cells increased 21-fold from 4.8±1.8 pre-immune to 105±24 after three immunizations and stayed constant throughout the trial. This increase in frequency is similar to that of all patients' CD8 cells when challenged with the untransfected wild type AD100 (FIG. 2D). Finally, the specificity of the response is evident from the absence of an increase of the response to K562 (FIG. 2E) or of unchallenged CD8 cells. The CD8 response to K562 and to AD100 in its w. t. form or after genetic modification is significantly different at each time point after vaccination (FIG. 2F).

The CD8 response listed in Table 2 reports the response to the matched vaccine for A1 or A2 positive patients. For non A1, A2 patients, it is the response to AD100-A2. One of 15 patients could not be analyzed due to renal failure unrelated to the trial prior to completing the first course of immunization. Of the fifteen patients treated, five patients had clinical responses: one partial response (PR), and four patients with stable disease (SD). Four of these patients with clinical responses, (PR+3 SD), are still alive with stabilization of their diseases without further therapy for: 31, 28, 25, and 12 months.

The patient that died, originally had SD for 5 months then progressed and died 15 months later in spite of several courses of palliative chemotherapy. In contrast, nine of the other ten patients that did not respond to the vaccination are deceased except one patient who achieved stable disease after therapy with Iressa™. Table 2 summarizes the data for all patients, including pre-trial treatment, clinical response to immunization and immune response. Patients that had progressive disease while under treatment went off study as indicated in Table 2.

Table 2 shows a summary of clinical responses, immunological CD8 responses, survival and pretreatment of fifteen patients with advanced stage IIIB/IV NSCLC treated with allogeneic B7/HLA A transfected NSCLC vaccine. The abbreviations in Table 2 are: PD—progressive disease; NE—not evaluable for immune response, but included in survival analysis on the right; PR—partial response; SD-sable disease; C—chemotherapy; R—radiation; S—surgery. Survival indicates time of survival since study entry; +indicates patient alive; n. d. no done, patients off study because of progression.

TABLE 2 Summary of Clinical Responses, Immunological CD8 Responses, Survival and Pretreatment of Fifteen NSCLC Patients. Ifn-γ producing CD8 cells to AD100-HLA Fold Time to challenge (spots per 20,000) Patient # Titer Previous Survival Progression Pre- 1st 2nd 3rd HLA Response increase TX (mos) (mos) immune course course course 1005 A1 PD 190 C + R 10 — 0 190 n.d. n.d. 1012 A1 NE NE C 15 — 0.2 n.d. n.d. n.d. 1001 A2 PD 25 C + S 18 — 0 25 n.d. n.d. 1002 A2 PD 1.6 C + S 22 — 41 65 n.d. n.d. 1009 A2 PD 6.5 C  3 — 2 13 n.d. n.d. 1010 A2 PR 41 S   27+ 3 3.8 46 88 157  1011 A2 PD 19 C 11 — 3 30 57 n.d. 1013 A2 PD 34 C + R + S  2 — 5.2 164 178 n.d. 1014 A2 SD 19 C + S   13+ 3 1.6 30 30 25 1015 A2 PD 0 C + R  7 — 0 0 nd nd 1003 non SD 134 S   31+ 26+ 1 134 113 84 1004 non SD 424 C + R 23 11   0 424 232 >450  1006 non PD 9.3 C + S   30+ — 16 150 n.d. n.d. 1007 non SD 14 C + R + S   29+ 23+ 1.2 2.8 .8 0/17 1008 non PD 32 C  6 — 5.6 178 n.d. n.d.

Five patients had a clinical response and the frequency of IFN-spot forming CD8 cells increased upon successive imnunization as measured by challenge ex vivo with transfected or untransfected AD100, while the reactivity to K562 remained low and unchanged (FIG. 2E). In three of the clinically responding patients (FIG. 2; 1004, 1007, 1010), blood samples were obtained after completion of the 18 week treatment period at 35 to 75 weeks post trial entry and showed still a considerable titer of CD8 cells responding to AD100 (FIG. 2G). Indeed, in two of two patients (1004, 1007), the titer increased further even after immunization was ended at 18 weeks.

The median survival time of all patients at the time of analysis was 18 months, exceeding the expected median survival time of less than one year for this group of patients (FIG. 3). 90% confidence intervals are shown in FIG. 3. Analysis of survival by MHC matching and by clinical response revealed that HLA unmatched patients showed a survival advantage that with p=0.07 was not statistically significant while clinical responders had a significant (p=0.008) survival advantage when compared to non responders.

Safety. None of the 15 patients entered into the trial experienced any treatment related serious adverse events, defined as deaths or events requiring hospitalization. Treatment related side effects consisted of local erythema and swelling that resolved in three to four days. One patient complained about transient arthralgias that may have been treatment related. One patient died within 30 days of the last immunization due to pulmonary failure; one patient who had previous episodes of pericarditis experienced pericardial effusion during the last course of immunization, requiring a pericardial window. No tumor cells were detected in the fluid; the patient responded to immunization and is still in stable disease. As mentioned above, one patient had renal failure prior to completion of one course of immunization. None of these events were deemed likely to be treatment related by an independent safety monitoring board.

Example 3 Further Characterization of Advanced Lung Cancer Patients to Whole Cell Immunization with an Allogeneic Vaccine

This example describes a continuation of the study described in Example 2, including additional patients and time of study. Experiments were performed essentially as described in Example 2 and Raez et al., J. Clin. Oncol. 22: 2800-2807 (2004).

Patient Characteristics. The characteristics of the 19 study patients are outlined in Table 3. Eastern Cooperative Oncology Group performance status was 0 to 1 in 18 patients (74%). Thirteen patients received vaccine matched for HLA, either A1 (three patients) or A2 (10 patients), whereas the six patients who were non-A1 and non-A2 received unmatched vaccine (that is, HLA-A1 vaccine). While HLA A matched patients may be able to mediate CD8 responses by direct antigen presentation by the vaccine cells, it was reasoned that unmatched patients may, nonetheless, mount a CD8 response via indirect antigen presentation after vaccine cell death and antigen uptake by antigen presenting cells. Before being enrolled on study, all patients had been previously treated: nine (47%) with surgery, six (32%) with radiation therapy, and 17 (89%) with chemotherapy. Among the chemotherapy-treated patients, 10 (53%) had been unsuccessfully treated with more than one chemotherapy regimen.

TABLE 3 Characteristics of the 19 patients enrolled in the study. Characteristic No. of Patients Age, years* <50 2 50-59 6 60-69 5 70+ 6 Sex Female 12 Male 7 Race/ethnicity White non-Hispanic 13 White Hispanic 5 Black non-Hispanic 1 Pathology Adenocarcinoma 11 Bronchoalveolar 3 Squamoous cell 3 Undifferentiated 2 Metastasis site Adrenal 1 Brain 3 Liver 1 Lung 9 Pleura 1 Multiple sites† 4 ECOG performance status 0 4 1 14 2 1 HLA A1 3 A2 10 Neither 6 Abbreviation: ECOG, Eastern Cooperative Oncology Goup. *Mean = 62 years; range 36 to 82 years. †One pancreas/lung/adrenal; one brain/lung; one lung/adrenal; one liver/lung/T-spine.

Clinical Outcomes. Eighteen patients received a total of 30 courses of vaccine, 90 vaccinations in total (Table 4). Five patients received three full courses, and two patients had two full courses. With the exception of one patient taken off study because a serious adverse event (SAE) occurred after the first vaccination (zero courses completed), the remaining 11 patients had one full course, after which they were taken off study because of disease progression. Four patients experienced SAEs after vaccination, none of which was judged to be vaccine-related.

TABLE 4 Outcomes in the 19 Patients Enrolled On Study. Outcome No. of Patients Courses of vaccine received 0 1 1 11 2 2 3 5 Clinical response Complete 0 Partial 1 Stable disease 5 Progressive disease 13 Serious AEs (grade 3 and 4) Pericardial effusion 2 Renal Failure 1 Respiratory failure 1 AEs (grade 1 or 2) Rash 1 Chest pain* 1 Joint pain 1 Status† Alive 7 Dead 12 Abbreviation: AE, adverse event. *Chest pain/shortness of breath. †Alive: median follow-up was 36 months (range, 10 to 40 months); time of death ranged from 1 to 23 months after entry on study.

During the first course of vaccination, a 58-year-old woman developed malignant pericardial effusion requiring a pericardial window; the patient was taken off study, discharged to hospice, and died 1 week later. She had previously been treated unsuccessfully with five lines of palliative chemotherapy before enrollment on study. A 76-year-old male patient also developed a pericardial effusion requiring a pericardial window, but review of prior scans revealed developing pericardial effusion before entry on study. This patient, who had received three courses of vaccine before the SAE developed, continues to have stable disease. He is currently alive and well after 31 months without any further therapy.

A 55-year-old male was found to have worsening of chemotherapy-induced renal dysfunction the day of his first vaccination after he had already signed consent 1 week earlier and underwent a preliminary skin test. His renal function continued deteriorating in the following days, and he died 3 months later. The fourth patient who experienced a SAE was a 56-year-old woman with brain metastasis. During her second course of vaccination, she developed respiratory failure, was then taken off study, and died within 30 days from progression of her disease. This patient had previously been unsuccessfully treated with four lines of palliative chemotherapy.

Regarding other side effects, one patient complained of transient pain at the injection site. Four patients developed some erythema at the vaccination site that resolved within a week. One patient experienced moderate arthritic pain in several joints after the first course. We did not find any patients with significant alteration of their laboratory parameters, including: complete blood and platelet counts, creatinine/BUN, calcium, and liver function tests. Table 5 shows time to response, duration of response, and survival time for the six patients who had response on study.

TABLE 5 Time to Response, Duration of Response, and Survival Time for the Six Patients Who Had Response on Study. Time to Duration of Survival Response Response Time Patient ID Response (months) (months) (months)* 1010 PR 2.3 13   36+ 1003 SD 1.9 39+  40+ 1004 SD 1.6 3.5 23   1007 SD 2.1 2.5 37+ 1014 SD 2.3 3.5 21+ 1016 SD 1.9 1.6 11+ Abbreviations: PR, partial response; SD, stable disease *Patients alive as of February, 2004 denoted by plus sign.

One patient had a partial response lasting 13 months, and five showed stable disease ranging from 1.6 to 39+ months (Table 5). The clinical response rate was 32% (six of 19 patients). As of February 2004, these patients had survival times ranging from 23 to 40+ months, and five patients were still alive.

After the patient who had a partial response developed new malignant lesions, verified by positron emission tomography scan, she was put under observation for 2 months because her disease was judged clinically nonaggressive. Several lesions subsequently decreased in size or disappeared. This patient continues to have stable disease without need of palliative chemotherapy 36 months after completing vaccination. Only one of the six patients who had a response on treatment required subsequent palliative chemotherapy. The remaining five patients continue to have stable disease without need of further treatment.

Among the other 13 patients who did not respond to therapy, only two were alive as of February 2004. One of these patients experienced disease stabilization with gefitinib (Iressa™), and the other is undergoing palliative chemotherapy.

Logistic regression analyses of age, sex, race. pathology, and HLA-matching of vaccine showed that none of these factors were statistically significantly related (P>0.10 in all instances) to clinical response (that is, to partial response or stable disease).

FIG. 4 shows the Kaplan-Meier estimate of overall survival for the 19 study patients (vertical tick marks indicate censored follow-up). The estimated median survival time is 18 months (90% CI, 7 to 23 months). Estimates of 1-year, 2-year, and 3-year overall survival are 52% (90% CI, 32% to 71%), 30% (90% CI, 11% to 49%), and 30% (90% CI, 11% to 49%), respectively. As of February 2004, death had occurred in 12 patients from 1 to 23 months after entry on study (Table 2). For the seven patients who are still alive, follow-up from study entry currently ranges from 10 to 40 months, with a median follow-up time of 36 months.

Univariate proportional hazards regression analysis suggested a possibly higher mortality rate in patients receiving HLA-matched vaccine (hazard ratio=4.5; 90% CI, 1.1 to 17.2), and a possibly lower mortality rate in patients with adenocarcinoma (hazard ratio=0.3; 90% CI, 0.1 to 1.0). A multivariate analysis involving five covariates (HLA-matching, age, sex, race, pathology), however, discounted an adverse effect of HLA-matching of vaccine on overall mortality; the corresponding adjusted hazard ratio was 1.9 (P=0.51). The adjusted hazard ratio for adenocarcinoma versus other pathologies was 0.2 (P=0.11), which is within the realm of chance at conventional levels of significance.

Immune Response to Vaccination. This cohort of patients had been heavily pretreated and carried large tumor burdens that are believed to be immunosuppressive. It was important, therefore, to establish whether the tumor vaccination protocol was able to induce a specific immune response in these patients. Since the CD8 CTL response is thought to be critical for tumor rejection, studies were focused on this arm of the immune system. To distinguish between nonspecific natural killer (NK) activity and CD8 CTL activity, a two-fold strategy was employed. First, CD8 cells were purified to eliminate NK cells by including anti-CD56 in the negative selection cocktail of antibodies. Second, the CD8 cells were challenged with K562, an NK target. NK contamination would result in high titers of cells responding to K562 challenge.

All but one patient had a measurable CD8 response after 6 weeks (three vaccinations) that tended to increase after 12 weeks and stabilize by 18 weeks (Table 6). In vitro challenge of patient CD8 cells with wild type A1 or A2 transfected AD100 did not reveal significant differences. Two patients (patient Nos. 1012 and 1019) could not be evaluated immunologically because there was no follow-up sample available for analysis due to early disease progression or adverse events. One patient had only a very modest response, while most other patients showed a strong, highly statistically significant response to vaccination (see pre- and postimmunization titers on challenge with vaccine cells, and lack of response to K562 control; FIG. 5, top panels). All but one patient had a measurable CD8 response after 6 weeks (three vaccinations) that tended to increase after 12 weeks and stabilize by IS weeks (Table 6). In vitro challenge of patient CD8 cells with wild type A1 or A2 transfected AD100 did not reveal significant differences. Two patients (patient Nos. 1012 and 1019) could not be evaluated immunologically because there was no follow-up sample available for analysis due to early disease progression or adverse events. One patient had only a very modest response, while most other patients showed a strong, highly statistically significant response to vaccination (see pre- and postimmunization titers on challenge with vaccine cells, and lack of response to K562 control; FIG. 5, top panels).

TABLE 6 CDB Response of Vaccinated Patients Immune Response of CDB Cells to Vaccination* 0 Weeks 6 Weeks 12 Weeks 18 Weeks HLA/Patient AD- AD- AD- AD- AD- AD- AD- AD- AD- AD- AD- AD- NO. wt A1 A2 K562 wt A1 A2 K562 wt A1 A2 K562 wt A1 A2 K562 A2/1001 4 6.2 0 2.6 51 49 25 6 A2/1002 12 19 41 170 30 55 65 96 NO/1003 1 1 7 0 70 134 53 0 31 113 27 0 49 84 23 6 NO/1004 0 0 0 5 321 424 195 0 216 232 150 0 283 450 130 0 A1/1005 15 0 0 40 92 190 80 34 NO/1006 13 17 12 11 156 152 132 16 NO/1007 0 1 0 0 0 3 0 0 1 1 1 0 0 0 2 0 NO/1008 5 6 4 10 97 180 48 3 A2/1009 3 4 2 17 13 39 13 18 A2/1010 8 8 4 14 48 87 46 5 120 163 88 8 185 241 157 17 A2/1011 14 20 3 15 80 150 30 12 88 226 57 4 A2/1013 18 150 5 0 155 300 164 3 175 154 178 3 A2/1014 3 2 2 10 28 20 30 9 30 20 30 12 25 23 25 4 A2/1015 0 0 0 0 0 0 0 0 A1/1016 138 120 128 4 144 150 163 5 127 120 164 15 A2/1017 0 11 0 4 100 200 200 3 NO/1018 13 44 0 9 51 200 52 9 NOTE. CD8 cells challenged at a ratio of 20:1 = CD8: tumor cell. The mean spot number of quadruplicate values is given. Abbreviations: AD-wt, AD100 untransfected; AD-A1 or AD-A2, AD100 transfected with HLA A1 or A2; HLA NO, No HLA A1 or A2. *Values are number of interferon-gamma secreting cells (spots per 20,000 CD8 cells) after in vitro challenge.

There was no statistically significant difference in the CD8 response depending on whether or not the patients were HLA-matched to the vaccine (Table 6). Most patients before vaccination had only low or absent immune response to vaccine cells, and equally low activity to challenge with K562. One patient (No. 1016) had strong prevaccination CD8 activity toward AD100 and only minimal activity toward K562 (FIG. 5, last panel), suggesting preexisting immune activity toward the tumor. Another patient (No. 1002) had high prevaccination K562 reactivity of his CD8 cells and low activity toward AD100. Vaccination increased reactivity toward AD100 and tended to decrease CD8 reactivity toward K562 when it was present.

The immune response of the six clinically-responding patients (FIG. 5B, lower panels) shows that CD8 titers to AD100 stimulation continue to be elevated up to 150 weeks after cessation of vaccination.

Given the advanced stage of disease in patients enrolled in the studies disclosed herein, the evidence of some clinical benefit was unexpected and encouraging. Moreover, since the B7-vaccine tested here induced CD8 CTL responses, it may be that the CD8 response is causally related to the clinical outcome seen here. Additional studies are performed in the setting of minimal disease. Patients with early stage NSCLC (stageI/II) are vaccinated after surgery to decrease the chance of relapse and potentially prolong survival.

The results described in this example show that tumor progression can be slowed by vaccination and that this effect occurs regardless of whether or not patients are allogeneic to the HLA A1 or A2 locus of the vaccine. These findings also support indirect antigen presentation as being effective in promoting antitumor activity and that allogeneic MHC molecules enhance the effect.

Example 4 Establishment and Expansion of AD100-A1-B7.1 Cells

A human lung adenocarcinoma cell line (designated AD100) was established in 1994 at the University of Miami, derived from a patient with NSCLC. This cell line has been kept in culture in standard medium and is free of contamination by Mycoplasma, virus, or other adventitious agents. It is homogeneous, adherent to plastic, and grows at a rate of division of approximately 26 hours.

AD100 cells are transfected with plasmid cDNA, pBMG-Neo-B7.1 and pBMG-His-HLA A2 or with B45-Neo-CM-A1-B7.1. Transfected cells were selected with G418 and Histidinol. Verification of correct sequences was based on restriction analysis and the expression of the relevant gene products, namely G418 or histidinol resistance for the vector sequence, HLA A1, A2, and B7.1 expression for the transfected cDNA. The minimum requirement for their use as vaccine was the coexpression of HLA A1 or A2 plus B7.1 on at least 70% of the cells as shown in FIG. 1 a for representative batches of vaccine cells.

AD100-A1-67.1 cells may be previously prepared and frozen in aliquots. The cryovial containing the cells is completely thawed rapidly using a 37° C. water bath and gentle swirls. The cells are then transferred the cells immediately to a previously prepared sterile 15 ml conical centrifuge tube kept on ice. To this 15 ml conical centrifuge tube, 9 ml of Complete Media 1 (IMDM; FBS Certified heat inactivated-final con. 9%; Gentamicin-final conc. 0.04 mg/ml) is slowly added 1 to 2 drops at a time, while gently swirling the tube in order to uniformly mix cells with media. This process should take 10 to 15 minutes. After all the media is added, the cells are centrifuged cells at 300×g (1200 rpm) for 10 minutes, at room temperature, with the brake set to “Low”. The supernatant is then gently aspirated away and the cells are resuspend in 10 ml of Complete Media 2 (IMDM; FBS Certified heat inactivated-final con. 9%; Gentamicin-final conc. 0.04 mg/ml; Geneticin G-418-final conc. 1 mg/ml), equilibrated to room temperature.

A cell count and viability test, using Trypan Blue @ 1:10 dilution is then performed. Cells are then seeded at 2×10⁶ cells per T-175 tissue culture flask containing 35 ml of Complete Media 2. The seeded flasks are then incubated for 3 to 5 days in a 37° C. incubator with 5% CO₂.

Feeding Cells for Working Cell Bank

The cells should not be disturb until the third day of culture, when an assessment of whether cells have attached to the flask should be made. On the 3rd day of culture, a percentage of cells that have attached needs to be estimated. If ≧70% of the cells have attached to the flask, the media needs to be changed. Old media should be removed using an aspirating pipette and 50 ml of fresh Complete Media 1 pre-warmed to 37° C. should be added to each flask. The flasks are then returned to 37° C. incubator with 5% CO₂ for further culture. If, when observing cells on the third day of culture, ≦70% of cells are deemed to be attached, they need to be left until the fifth day without changing media. After 3-5 days in culture, remove flasks from the 37° C. incubator and determine percentage of confluency. Cells need to be cultured until such time when they are deemed to be 90-95% confluent. The cells must be split when the confluency reaches 90-95% per flask.

Harvesting the Cells with Trypsin EDTA for Working Cell Bank

After the cells reach 90-95% confluency, the cells are harvested by aspirating off the supernatant and by adding 12 ml of Trypsin-EDTA pre-warmed to 37° C. to each flask. The cells are incubated at 37° C. in this solution for approximately 20 minutes. After incubation, the flask is vigorously shaken across its surface area, to ensure that the cells are no longer adhering to the flask. 13 ml of Complete is then added to neutralize Trypsin-EDTA reaction. The supernatant containing the cells that have detached from the flask is then collected and transferred it to a sterile 50 ml or 250 ml conical centrifuge tube. Cell suspensions from all the flasks should be combined and washed at the same time. The cells are then centrifuged at 300×g (1200 rpm) for 10 minutes, at room temperature with the brake set to “Low”. The supernatant is then aspirated off and the cells are resuspend in 15-30 ml of pre-warmed (to 37° C.) Complete Media 2.

A cell count and viability test using Trypan Blue 1:10 dilution is them performed.

New T-175 tissue culture flasks are then seeded at the density of 2.0×10⁶ cells per flask, using pre-warmed (to 37° C.) Complete Media 2. The total volume of the Complete Media 2 in each T-175 tissue culture flask should be 35 ml.

The above harvesting and expanding process is repeated approximately every 7 days until 201 T-175 flasks can be seeded at one time. When this threshold is met, Complete Medium 2 is used to seed the cells for the final expansion. After the first 3-5 days of culture, when the cells have attached and are ready to be fed, change to Complete Medium 1 is used. When the cells reach 90-95% confluency, the cells are harvested as above. The cells are then washed twice in at least 200 ml of (4° C.) Wash Media (0.9% sodium chloride; 0.5% HAS; and 0.0067% USP sodium bicarbonate). After the second wash, the cell pellet is resuspend in Wash Media to the final volume of 200 ml. A cell count and viability test using 1:70 dilution of Trypan Blue is again performed. The cells are then irradiated at 12,000 rads using a Cobalt irradiator. The cells are now ready for cryopreservation.

Cryopreservation of Expanded AD100-A1-B7.1 Cells

At least 80-120 cryovials should be labeled with cell identification, batch number, cell concentration, tech's initials, and date. The cells are then centrifuged at 4° C., 300×g (1200 rpm), for 10 minutes, with the brakes on. After which, the supernatant is aspirated off and the pelleted cells are placed on ice. The cells are then resuspend slowly with gentle mixing, to a concentration of 200×10⁶/ml ice cold Wash Media. Ice cold Freezing Media (0.9% sodium chloride; 0.5% HAS; 0.0067% USP sodium bicarbonate; and 20% DMSO) is slowly added at a 1:1 ratio to have a cell concentration of 100×10⁶/ml and DMSO concentration of 10%. The cells are then aliquoted at 0.5 ml (50×10⁶ cells) previously prepared cryovials on ice and then stored at −80° C. for 18-24 hours. After 24 hours, the frozen cells are transferred to the Liquid Nitrogen storage tank.

Throughout this application various publications have been referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains to the same extent as if each was specifically and individually indicated to be incorporated by reference. The patents, published applications, and scientific literature referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.

Example 5 1. Phase 1 Trial Design and Results

Three vaccinations, each of which was spaced 2 weeks apart, comprised one course of treatment. At the end of the first course, patients who had evidence of stable disease or responding NSCLC (by computed tomography scans), and no to moderate toxicity (grade≦2), were treated with a second course of vaccination. No patient was denied a second or third course of treatment because of toxicity.

In the absence of tumor progression or severe toxicity (grade≧3), a third course of vaccination was given. No patients experienced drug-related toxicity of grade≧3, and so all patients who did not progress were eligible for the third course of vaccination.

Therefore, a total of three courses, or nine total vaccinations, were possible in the study. Clinical and toxicity evaluations were done before and after each vaccination, and immunologic assessment was made before and after each course.

A. Survival Status and History of all Patients Tested in Trials.

The up-to-date survival curve is presented in FIG. 7. The full patient history and follow up is presented in Table 7.

B. Current Status of Responders.

Of six clinical responders, three have since died, the most recent in February 2007 (patient # 14 below, and in Table 1). As of March 2007, there are three continuing survivors. The mean survival of the six clinical responders is currently 59+ months (median=˜66+ (60 or 72+) months). The detailed status of the original six clinical responders is shown below (Patient # references to Table 7):

TABLE 7 Patient # Status Survival 4 Dead 23 mos. 16 Alive 48+ mos.   14 Dead 60 mos. 10 Alive 72+ mos.   7 Dead 75 mos. 3 Alive 76+ mos.  

C. Up-to-Date Survival Curve (from Initiation of Trial to Present or to Last Survivor) Please see FIG. 7.

D. Percentage of the Patients Responded or Had an Adverse Effect.

As seen in Table 8, 19 patients were enrolled into the trial. It should be noted that one patient was taken off study before receiving any vaccinations, but he is still counted among the 19 patients.

Six of the 19 patients (32%) responded clinically with either partial response (PR) or stable disease (SD).

Three of the 19 patients (16%) experienced adverse events (grade 1 or 2) which were judged to be potentially vaccine-related. These adverse events were comprised of: rash (1 patient), moderate arthritic pain in the joints (1 patient), and chest pain (1 patient). Additionally, four of the 19 patients (21%) developed some transient erythema at the vaccination site. The erythema is not considered to be an adverse event since it resolved within a week.

None of the 19 patients (0%) experienced drug-related serious adverse events (SAEs). All SAEs were judged to be not vaccine-related. Four of the 19 patients (21%) experienced non-drug-related SAEs.

TABLE 8 HLA Location of Number of Clinical Additional Survival Patient Sex Type Pathology Metastasis Vaccinations Response Chemotherapy (weeks) AE or SAE 1 F A2 A B 3 PD None 26 Rash 2 F A2 A L 3 PD Iressa 43 None 3 F Non A P, L, A 9 SD None   328++ None 4 F Non A B 9 SD Gemzar; 100  None Carboplatin 5 M A1 B L 3 PD None 34 None 6 M Non A B, L 3 PD Iressa 223  None Temodar 7 M Non A L 8, SD Revaccinated with 324  Pericardial 9 PT 107; Effussion* Tarceva (1 yr. later) (recurrent) 8 F Non A L 3 PD Gemzar 26 None 9 M A2 U L 3 PD None 12 None 10 F A2 A L, A 9 PR None   312++ Joint pain 11 M A2 S L 3 PD None 45 Chest pain 12 M A1 S A 8 PD None 13 Renal Failure* 13 F A2 A B 4 PD None 11 Respiratory Failure* 14 F A2 S L 9 SD Iressa; 238† None Tarceva 15 F A2 A L 3 PD Iressa 30 None 16 F A1 B L 6 SD Velcade;   207++ None Alimta; Iressa 17 F A2 B Pl 3 PD Gemzar; 23 None Navelbine 18 M Non B Li, L, T 3 PD None 49 None 19 F A2 U M, Li 1 PD None  4 Pericardial Effussion* (tumor) Notes: Follow up as of Feb. 21, 2007. ++Still alive as of February 2007. †Died February 2007. *Adverse event was determined to be not related to vaccine. Sex: F = female M = male HLA Type: A1 = HLA A1 A2 = HLA A2 Non = Non-HLA A1/A2 Pathology: A = adenocarcinoma B = bronchial carcinoma S = squamous cell carcinoma U - undifferentiated carcinoma Location of Metastasis: B = brain L = lung A = adrenal gland P = pancreas Pl = pleura Li = liver T = thoracic spinal column Clinical Response (responders shown in red): PD = progressive disease SD = stable disease PR = partial response

E. Patients Response to Different Levels of Vaccine.

Please see the graph in FIG. 8 for further detail. Patients who received a second or third course of vaccination fared much better in terms of both clinical response and survival. All five patients who received 8 or 9 vaccinations were clinical responders. Of the clinical responders, 5 of 6 (83%) received 8 or 9 vaccinations in their initial therapy. Of the non-responders, 12 of 13 (92%) received 0-3 vaccinations.

F. Responder Breakdown Regarding Pathology Of NSCLC Cell Type (Adenocarcinoma, Bronchoalveolar, Squamous and Undifferentiated).

Of the 6 clinical responders, the pathology was as follows: 4 had adenocarcinoma, 1 had bronchoalveolar carcinoma, and 1 had squamous cell carcinoma. On a percentage basis, 4 of 11 (36%) patients with adenocarcinoma responded, 1 of 3 (33%) patients with bronchoalveolar carcinoma responded, 1 of 3 (33%) patients with squamous cell carcinoma responded, and 0 of 2 (0%) patients with undifferentiated carcinoma responded. Please see Table 1 for further detail.

G. Comparison of Matched and Non-Matched HLA in Trials and Patients.

A multivariate analysis involving five covariates (HLA-matching, sex, race, pathology) showed no statistical significance of HLA-matching on overall mortality.

Of the 19 patients, 13 were matched (3 at A1, 10 at A2), and 6 were non-matched. Of the 6 clinical responders, 3 were HLA matched, and 3 were non-matched. Among matched patients, 1 of the 3 (33%) A1-matched patients were clinical responders, and 2 of 10 (20%) A2-matched patients were clinical responders. Among non-matched patients, 3 of 6 (50%) were clinical responders.

It should be noted that logistic regression analyses of age, sex, race, pathology, and HLA-matching of vaccine showed that none of these factors were statistically significantly related (P≧0.10 in all instances) to clinical response.

Univariate proportional hazards regression analysis suggested a possibly higher mortality rate in patients receiving HLA-matched vaccine (hazard ratio=4.5; 90% CI, 1.1 to 17.2), and a possibly lower mortality rate in patients with adenocarcinoma (hazard ratio=0.3; 90% CI, 0.1 to 1.0). A multivariate analysis involving five covariates (HLA-matching, age, sex, race, pathology) however, discounted an adverse effect of HLA-matching of vaccine on overall mortality; the corresponding adjusted hazard ratio was 1.9 (P=0.51). The adjusted hazard ratio for adenocarcinoma versus other pathologies was 0.2 (P=0.11), which is within the realm of chance at conventional levels of significance.

BPV-1-B7.1-HLA A1 vector sequence XbaT SEQ ID NO:1     ~~~~~~     1 TCTAGAGAGC TTGGCCCATT GCATACGTTG TATCCATATC ATAATATGTA AGATCTCTCG AACCGGGTAA CGTATGCAAC ATAGGTATAG TATTATACAT    51 CATTTATATT GGCTCATGTC CAACATTACC GCCATGTTGA CATTGATTAT GTAAATATAA CCGAGTACAG GTTGTAATGG CGGTACAACT GTAACTAATA   101 TGACTAGTTA TTAATAGTAA TCAATTACGG GGTCATTAGT TCATAGCCCA ACTGATCAAT AATTATCATT AGTTAATGCC CCAGTAATCA AGTATCGGGT   151 TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ATATACCTCA AGGCGCAATG TATTGAATGC CATTTACCGG GCGGACCGAC   201 ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA TGGCGGGTTG CTGGGGGCGG GTAACTGCAG TTATTACTGC ATACAAGGGT   251 TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA ATCATTGCGG TTATCCCTGA AAGGTAACTG CAGTTACCCA CCTCATAAAT   301 CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCATTTGAC GGGTGAACCG TCATGTAGTT CACATAGTAT ACGGTTCATG   351 GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC CGGGGGATAA CTGCAGTTAC TGCCATTTAC CGGGCGGACC GTAATACGGG   401 ACTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA TCATGTACTG GAATACCCTG AAAGGATGAA CCGTCATGTA GATGCATAAT   451 GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG CAGTAGCGAT AATGGTACCA CTACGCCAAA ACCGTCATGT AGTTACCCGC   501 TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG ACCTATCGCC AAACTGAGTG CCCCTAAAGG TTCAGAGGTG GGGTAACTGC   551 TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT AGTTACCCTC AAACAAAACC GTGGTTTTAG TTGCCCTGAA AGGTTTTACA   601 CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GCATTGTTGA GGCGGGGTAA CTGCGTTTAC CCGCCATCCG CACATGCCAC   651 GGAGGTCTAT ATAAGCAGAG CTCGTTTAGT GAACCGTCAG ATCGCCTGGA CCTCCAGATA TATTCGTCTC GAGCAAATCA CTTGGCAGTC TAGCGGACCT   701 GACGCCATCC ACGCTGTTTT GACCTCCATA GAAGACACCG GGACCGATCC CTGCGGTAGG TGCGACAAAA CTGGAGGTAT CTTCTGTGGC CCTGGCTAGG   751 AGCCTCCGGT CGATCGACCG ATCCTGAGAA CTTCAGGGTG AGTTTGGGGA TCGGAGGCCA GCTAGCTGGC TAGGACTCTT GAAGTCCCAC TCAAACCCCT   801 CCCTTGATTG TTCTTTCTTT TTCGCTATTG TAAAATTCAT GTTATATGGA GGGAACTAAC AAGAAAGAAA AAGCGATAAC ATTTTAAGTA CAATATACCT   851 GGGGGCAAAG TTTTCAGGGT GTTGTTTAGA ATGGGAAGAT GTCCCTTGTA CCCCCGTTTC AAAAGTCCCA CAACAAATCT TACCCTTCTA CAGGGAACAT   901 TCACCATGGA CCCTCATGAT AATTTTGTTT CTTTCACTTT CTACTCTGTT AGTGGTACCT GGGAGTACTA TTAAAACAAA GAAAGTGAAA GATGAGACAA   951 GACAACCATT GTCTCCTCTT ATTTTCTTTT CATTTTCTGT AACTTTTTCG CTGTTGGTAA CAGAGGAGAA TAAAAGAAAA GTAAAAGACA TTGAAAAAGC  1001 TTAAACTTTA GCTTGCATTT GTAACGAATT TTTAAATTCA CTTTTGTTTA AATTTGAAAT CGAACGTAAA CATTGCTTAA AAATTTAAGT GAAAACAAAT  1051 TTTGTCAGAT TGTAAGTACT TTCTCTAATC ACTTTTTTTT CAAGGCAATC AAACAGTCTA ACATTCATGA AAGAGATTAG TGAAAAAAAA GTTCCGTTAG  1101 AGGGTATATT ATATTGTACT TCAGCACAGT TTTAGAGAAC AATTGTTATA TCCCATATAA TATAACATGA AGTCGTGTCA AAATCTCTTG TTAACAATAT  1151 ATTAAATGAT AAGGTAGAAT ATTTCTGCAT ATAAATTCTG GCTGGCGTGG TAATTTACTA TTCCATCTTA TAAAGACGTA TATTTAAGAC CGACCGCACC  1201 AAATATTCTT ATTGGTAGAA ACAACTACAC CCTGGTCATC ATCCTGCCTT TTTATAAGAA TAACCATCTT TGTTGATGTG GGACCAGTAG TAGGACGGAA  1251 TCTCTTTATG GTTACAATGA TATACACTGT TTGAGATGAG GATAAAATAC AGAGAAATAC CAATGTTACT ATATGTGACA AACTCTACTC CTATTTTATG  1301 TCTGAGTCCA AACCGGGCCC CTCTGCTAAC CATGTTCATG CCTTCTTCTC AGACTCAGGT TTGGCCCGGG GAGACGATTG GTACAAGTAC GGAAGAAGAG  1351 TTTCCTACAG CTCCTGGGCA ACGTGCTGGT TGTTGTGCTG TCTCATCATT AAAGGATGTC GAGGACCCGT TGCACGACCA ACAACACGAC AGAGTAGTAA                 XhoI        Start B7.1 (CD80)          SEQ ID NO: 2       MetGly HisThrArg ArgGlnGly                ~~~~~~~      ~~~~~~~~~~~~~~~~~~~~~~~~~~  1401 TTGGCAAAGA ATTCCTCGAG GAAGCCATGG GCCACACACG GAGGCAGGGA AACCGTTTCT TAAGGAGCTC CTTCGGTACC CGGTGTGTGC CTCCGTCCCT ThrSerProSer LysCysPro TyrLeuAsn PhePheGlnLeu LeuValLeu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1451 ACATCACCAT CCAAGTGTCC ATACCTCAAT TTCTTTCAGC TCTTGGTGCTG TGTAGTGGTA GGTTCACAGG TATGGAGTTA AAGAAAGTCG AGAACCACGA AlaGlyLeu SerHisPheCys SerGlyVal IleHisVal ThrLysGluVal ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1501 GCTGGTCTT TCTCACTTCT GTTCAGGTGT TATCCACGTG ACCAAGGAAG CCGACCAGAA AGAGTGAAGA CAAGTCCACA ATAGGTGCAC TGGTTCCTTC *VLysGluVal AlaThrLeu SerCysGlyHis AsnValSer ValGluGlu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1551 TGAAAGAAGT GGCAACGCTG TCCTGTGGTC ACAATGTTTC TGTTGAAGAG ACTTTCTTCA CCGTTGCGAC AGGACACCAG TGTTACAAAG ACAACTTCTC LeuAlaGlnThr ArgIleTyr TrpGlnLys GluLysLysMet ValLeuThr* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1601 CTGGCACAAA CTCGCATCTA CTGGCAAAAG GAGAAGAAAA TGGTGCTGAC GACCGTGTTT GAGCGTAGAT GACCGTTTTC CTCTTCTTTT ACCACGACTG *MetMetSer GlyAspMetAsn IleTrpPro GluTyrLys AsnArgThrIle* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1651 TATGATGTCT GGGGACATGA ATATATGGCC CGAGTACAAG AACCGGACCA ATACTACAGA CCCCTGTACT TATATACCGG GCTCATGTTC TTGGCCTGGT *IPheAspIle ThrAsnAsn LeuSerIleVal IleLeuAla LeuArgPro ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1701 TCTTTGATAT CACTAATAAC CTCTCCATTG TGATCCTGGC TCTGCGCCCA AGAAACTATA GTGATTATTG GAGAGGTAAC ACTAGGACCG AGACGCGGGT SerAspGluGly ThrTyrGlu CysValVal LeuLysTyrGlu LysAspAla* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1751 TCTGACGAGG GCACATACGA GTGTGTTGTT CTGAAGTATG AAAAAGACGC AGACTGCTCC CGTGTATGCT CACACAACAA GACTTCATAC TTTTTCTGCG *PheLysArg GluHisLeuAla GluValThr LeuSerVal LysAlaAspPhe* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1801 TTTCAAGCGG GAACACCTGG CTGAAGTGAC GTTATCAGTC AAAGCTGACT AAAGTTCGCC CTTGTGGACC GACTTCACTG CAATAGTCAG TTTCGACTGA *PProThrPro SerIleSer AspPheGluIle ProThrSer AsnIleArg ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1851 TCCCTACACC TAGTATATCT GACTTTGAAA TTCCAACTTC TAATATTAGA AGGGATGTGG ATCATATAGA CTGAAACTTT AAGGTTGAAG ATTATAATCT ArgIleIleCys SerThrSer GlyGlyPhe ProGluProHis LeuSerTrp* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1901 AGGATAATTT GCTCAACCTC TGGAGGTTTT CCAGAGCCTC ACCTCTCCTG TCCTATTAAA CGAGTTGGAG ACCTCCAAAA GGTCTCGGAG TGGAGAGGAC *LeuGluAsn GlyGluGluLeu AsnAlaIle AsnThrThr ValSerGlnAsp* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1951 GTTGGAAAAT GGAGAAGAAT TAAATGCCAT CAACACAACA GTTTCCCAAG CAACCTTTTA CCTCTTCTTA ATTTACGGTA GTTGTGTTGT CAAAGGGTTC *AProGluThr GluLeuTyr AlaValSerSer LysLeuAsp PheAsnMet ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2001 ATCCTGAAAC TGAGCTCTAT GCTGTTAGCA GCAAACTGGA CTTCAATATG TAGGACTTTG ACTCGAGATA CGACAATCGT CGTTTGACCT GAAGTTATAC ThrThrAsnHis SerPheMet CysLeuIle LysTyrGlyHis LeuArgVal* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2051 ACAACCAACC ACAGCTTCAT GTGTCTCATC AAGTATGGAC ATTTAAGAGT TGTTGGTTGG TGTCGAAGTA CACAGACTAG TTCATACCTG TAAATTCTCA *AsnGlnThr PheAsnTrpAsn ThrThrLys GlnGluHis PheProAspAsn* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2101 GAATCAGACC TTCAACTGGA ATACAACCAA GCAAGAGCAT TTTCCTGATA CTTAGTCTGG AAGTTGACCT TATGTTGGTT CGTTCTCGTA AAAGGACTAT *ALeuLeuPro SerTrpAla IleThrLeuIle SerValAsn GlyIlePhe ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2151 ACCTGCTCCC ATCCTGGGCC ATTACCTTAA TCTCAGTAAA TGGAATTTTT TGGACGAGGG TAGGACCCGG TAATGGAATT AGAGTCATTT ACCTTAAAAA ValIleCysCys LeuThrTyr CysPheAla ProArgCysArg GluArgArg* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2201 GTGATATGCT GCCTGACCTA CTGCTTTGCC CCAAGATGCA GAGAGAGAAG CACTATACGA CGGACTGGAT GACGAAACGG GGTTCTACGT CTCTCTCTTC *ArgAsnGlu ArgLeuArgArg GluSerVal ArgProVal *** ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Stop  2251 GAGGAATGAG AGATTGAGAA GGGAAAGTGT ACGCCCTGTA TAACAGACTA CTCCTTACTC TCTAACTCTT CCCTTTCACA TGCGGGACAT ATTGTCTGAT                                                   XhoI                                                   ~~~~  2301 GTCAAATTAA GCCGAATTCT GCAGATATCC ATCACACTGG CGGCCGCTCG CAGTTTAATT CGGCTTAAGA CGTCTATAGG TAGTGTGACC GCCGGCGAGC XhoI ~~  2351 AGGAATTCAC TCCTCAGGTG CAGGCTGCCT ATCAGAAGGT GGTGGCTGGT TCCTTAAGTG AGGAGTCCAC GTCCGACGGA TAGTCTTCCA CCACCGACCA  2401 GTGGCCAATG CCCTGGCTCA CAAATACCAC TGAGATCTTT TTCCCTCTGC CACCGGTTAC GGGACCGAGT GTTTATGGTG ACTCTAGAAA AAGGGAGACG  2451 CAAAAATTAT GGGGACATCA TGAAGCCCCT TGAGCATCTG ACTTCTGGCT GTTTTTAATA CCCCTGTAGT ACTTCGGGGA ACTCGTAGAC TGAAGACCGA  2501 AATAAAGGAA ATTTATTTTC ATTGCAATAG TGTGTTGGAA TTTTTTGTGT TTATTTCCTT TAAATAAAAG TAACGTTATC ACACAACCTT AAAAAACACA  2551 CTCTCACTCG GAAGGACATA TGGGAGGGCA AATCATTTAA AACATCAGAA GAGAGTGAGC CTTCCTGTAT ACCCTCCCGT TTAGTAAATT TTGTAGTCTT  2601 TGAGTATTTG GTTTAGAGTT TGGCAACATA TGCCCATATG CTGGCTGCCA ACTCATAAAC CAAATCTCAA ACCGTTGTAT ACGGGTATAC GACCGACGGT  2651 TGAACAAAGG TTGGCTATAA AGAGGTCATC AGTATATGAA ACAGCCCCCT ACTTGTTTCC AACCGATATT TCTCCAGTAG TCATATACTT TGTCGGGGGA  2701 GCTGTCCATT CCTTATTCCA TAGAAAAGCC TTGACTTGAG GTTAGATTTT CGACAGGTAA GGAATAAGGT ATCTTTTCGG AACTGAACTC CAATCTAAAA  2751 TTTTATATTT TGTTTTGTGT TATTTTTTTC TTTAACATCC CTAAAATTTT AAAATATAAA ACAAAACACA ATAAAAAAAG AAATTGTAGG GATTTTAAAA  2801 CCTTACATGT TTTACTAGCC AGATTTTTCC TCCTCTCCTG ACTACTCCCA GGAATGTACA AAATGATCGG TCTAAAAAGG AGGAGAGGAC TGATGAGGGT                                           BamHI                                           ~~~~~~~  2851 GTCATAGCTG TCCCTCTTCT CTTATGGAGA TCCCTCGACG GATCCCTAGA CAGTATCGAC AGGGAGAAGA GAATACCTCT AGGGAGCTGC CTAGGGATCT  2901 GTCGAGGCGA TGCGGCGCAG CACCATGGCC TGAAATAACC TCTGAAAGAG CAGCTCCGCT ACGCCGCGTC GTGGTACCGG ACTTTATTGG AGACTTTCTC  2951 GAACTTGGTT AGGTACCTTG GTTTTTAAAA CCAGCCTGGA GTAGAGCAGA CTTGAACCAA TCCATGGAAC CAAAAATTTT GGTCGGACCT CATCTCGTCT  3001 TGGGTTAAGG TGAGTGACCC CTCAGCCCTG GACATTCTTA GATGAGCCCC ACCCAATTCC ACTCACTGGG GAGTCGGGAC CTGTAAGAAT CTACTCGGGG  3051 CTCAGGAGTA GAGAATAATG TTGAGATGAG TTCTGTTGGC TAAAATAATC GAGTCCTCAT CTCTTATTAC AACTCTACTC AAGACAACCG ATTTTATTAG  3101 AAGGCTAGTC TTTATAAAAC TGTCTCCTCT TCTCCTAGCT TCGATCCAGA TTCCGATCAG AAATATTTTG ACAGAGGAGA AGAGGATCGA AGCTAGGTCT  3151 GAGAGACCTG GGCGGAGCTG GTCGCTGCTC AGGAACTCCA GGAAAGGAGA CTCTCTGGAC CCGCCTCGAC CACCGACGAG TCCTTGAGGT CCTTTCCTCT  3201 AGCTGAGGTT ACCACGCTGC GAATGGGTTT ACGGAGATAG CTGGCTTTCC TCGACTCCAA TGGTGCGACG CTTACCCAAA TGCCTCTATC GACCGAAAGG  3251 GGGGTGAGTT CTCGTAAACT CCAGAGCAGC GATAGGCCGT AATATCGGGG CCCCACTCAA GAGCATTTGA GGTCTCGTCG CTATCCGGCA TTATAGCCCC  3301 AAAGCACTAT AGGGACATGA TGTTCCACAC GTCACATGGG TCGTCCTATC TTTCGTGATA TCCCTGTACT ACAAGGTGTG CAGTGTACCC ACCAGGATAG  3351 CGAGCCAGTC GTGCCAAAGG GGCGGTCCCG CTGTGCACAC TGGCGCTCCA GCTCGGTCAG CACGGTTTCC CCGCCAGGGC GACACGTGTG ACCGCGAGGT  3401 GGGAGCTCTG CACTCCGCCC GAAAAGTGCG CTCGGCTCTG CCAGGACGCG CCCTCGAGAC GTGAGGCGGG CTTTTCACGC GAGCCGAGAC GGTCCTGCGC  3451 GGGCGCGTGA CTATGCGTGG GCTGGAGCAA CCGCCTGCTG GGTGCAAACC CCCGCGCACT GATACGCACC CGACCTCGTT GGCGGACGAC CCACGTTTGG  3501 CTTTGCGCCC GGACTCGTCC AACGACTATA AAGAGGGCAG GCTGTCCTCT GAAACGCGGG CCTGAGCAGG TTGCTGATAT TTCTCCCGTC CGACAGGAGA  3551 AAGCGTCACC ACGACTTCAA CGTCCTGAGT ACCTTCTCCT CACTTACTCC TTCGCAGTGG TGCTGAAGTT GCAGGACTCA TGGAAGAGGA GTGAATGAGG                                     SalI                                    ~~~~~~  3601 GTAGCTCCAG CTTCACCACC AAGCTCCTCG ACGTCGACCC CAGACGCCGA CATCGAGGTC GAAGTGGTGG TTCGAGGAGC TGCAGCTGGG GTCTGCGGCT Start SEQ ID NO: 3  ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ HLA A1    MetAlaVal MetAlaPro ArgThrLeuLeu LeuLeuLeu SerGlyAla  3651 GGATGGCCGT CATGGCGCCC CGAACCCTCC TCCTGCTACT CTCGGGGGCC CCTACCGGCA GTACCGCGGG GCTTGGGAGG AGGACGATGA GAGCCCCCGG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ LeuAlaLeuThr GlnThrTrp AlaGlySer HisSerMetArg TyrPhePhe*  3701 CTGGCCCTGA CCCAGACCTG GGCGGGCTCC CACTCCATGA GGTATTTCTT GACCGGGACT GGGTCTGGAC CCGCCCGAGG GTGAGGTACT CCATAAAGAA ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *ThrSerVal SerArgProGly ArgGlyGlu ProArgPhe IleAlaValGly*  3751 CACATCCGTG TCCCGGCCCG GCCGCGGGGA GCCCCGCTTC ATCGCCGTGG GTGTAGGCAC AGGGCCGGGC CGGCGCCCCT CGGGGCGAAG TAGCGGCACC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *GTyrValAsp AspThrGln PheValArgPhe AspSerAsp AlaAlaSer  3801 GCTACGTGGA CGACACGCAG TTCGTGCGGT TCGACAGCGA CGCCGCGAGC CGATGCACCT GCTGTGCGTC AAGCACGCCA AGCTGTCGCT GCGGCGCTCG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ GlnLysMetGlu ProArgAla ProTrpIle GluGlnGluGly ProGluTyr*  3851 CAGAAGATGG AGCCGCGGGC GCCGTGGATA GAGCAGGAGG GGCCGGAGTA GTCTTCTACC TCGGCGCCCG CGGCACCTAT CTCGTCCTCC CCGGCCTCAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *TrpAspGln GluThrArgAsn MetLysAla HisSerGln ThrAspArgAla*  3901 TTGGGACCAG GAGACACGGA ATATGAAGGC CCACTCACAG ACTGACCGAG AACCCTGGTC CTCTGTGCCT TATACTTCCG GGTGAGTGTC TGACTGGCTC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *AAsnLeuGly ThrLeuArg GlyTyrTyrAsn GlnSerGlu AspGlySer  3951 CGAACCTGGG GACCCTGCGC GGCTACTACA ACCAGAGCGA GGACGGTTCT GCTTGGACCC CTGGGACGCG CCGATGATGT TGGTCTCGCT CCTGCCAAGA ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ HisThrIleGln IleMetTyr GlyCysAsp ValGlyProAsp GlyArgPhe  4001 CACACCATCC AGATAATGTA TGGCTGCGAC GTGGGGCCGG ACGGGCGCTT GTGTGGTAGG TCTATTACAT ACCGACGCTG CACCCCGGCC TGCCCGCGAA ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LeuArgGly TyrArgGlnAsp AlaTyrAsp GlyLysAsp TyrIleAlaLeu*  4051 CCTCCGCGGG TACCGGCAGG ACGCCTACGA CGGCAAGGAT TACATCGCCC GGAGGCGCCC ATGGCCGTCC TGCGGATGCT GCCGTTCCTA ATGTAGCGGG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LAsnGluAsp LeuArgSer TrpThrAlaAla AspMetAla AlaGlnIle  4101 TGAACGAGGA CCTGCGCTCT TGGACCGCGG CGGACATGGC GGCTCAGATC ACTTGCTCCT GGACGCGAGA ACCTGGCGCC GCCTGTACCG CCGAGTCTAG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ThrLysArgLys TrpGluAla AlaHisAla AlaGluGlnArg ArgValTyr*  4151 ACCAAGCGCA AGTGGGAGGC GGCCCATGCG GCGGAGCAGC GGAGAGTCTA TGGTTCGCGT TCACCCTCCG CCGGGTACGC CGCCTCGTCG CCTCTCAGAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LeuAspGly ArgCysValAsp GlyLeuArg ArgTyrLeu GluAsnGlyLys*  4201 CCTGGATGGC CGGTGCGTGG ACGGGCTCCG CAGATACCTG GAGAACGGGA GGACCTACCG GCCACGCACC TGCCCGAGGC GTCTATGGAC CTCTTGCCCT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LGluThrLeu GlnArgThr AspProProLys ThrHisMet ThrHisHis  4251 AGGAGACGCT GCAGCGCACG GACCCCCCCA AGACACATAT GACCCACCAC TCCTCTGCGA CGTCGCGTGC CTGGGGGGGT TCTGTGTATA CTGGGTGGTG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ProIleSerAsp HisGluAla ThrLeuArg CysTrpAlaLeu GlyPheTyr*  4301 CCCATCTCTG ACCATGAGGC CACCCTGAGG TGCTGGGCCC TGGGCTTCTA GGGTAGAGAC TGGTACTCCG GTGGGACTCC ACGACCCGGG ACCCGAAGAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *ProAlaGlu IleThrLeuThr TrpGlnArg AspGlyGlu AspGlnThrGln*  4351 CCCTGCGGAG ATCACACTGA CCTGGCAGCG GGATGGGGAG GACCAGACCC GGGACGCCTC TAGTGTGACT GGACCGTCGC CCTACCCCTC CTGGTCTGGG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *GAspThrGlu LeuValGlu ThrArgProAla GlyAspGly ThrPheGln  4401 AGGACACGGA GCTCGTGGAG ACCAGGCCTG CAGGGGATGG AACCTTCCAG TCCTGTGCCT CGAGCACCTC TGGTCCGGAC GTCCCCTACC TTGGAAGGTC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ LysTrpAlaAla ValValVal ProSerGly GluGluGlnArg TyrThrCys*  4451 AAGTGGGCGG CTGTGGTGGT GCCTTCTGGA GAGGAGCAGA GATACACCTG TTCACCCGCC GACACCACCA CGGAAGACCT CTCCTCGTCT CTATGTGGAC *HisValGln HisGluGlyLeu ProLysPro LeuThrLeu ArgTrpGluLeu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  4501 CCATGTGCAG CATGAGGGTC TGCCCAAGCC CCTCACCCTG AGATGGGAGC GGTACACGTC GTACTCCCAG ACGGGTTCGG GGAGTGGGAC TCTACCCTCG *LSerSerGln ProThrIle ProIleValGly IleIleAla GlyLeuVal ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  4551 TGTCTTCCCA GCCCACCATC CCCATCGTGG GCATCATTGC TGGCCTGGTT ACAGAAGGGT CGGGTGGTAG GGGTAGCACC CGTAGTAACG ACCGGACCAA LeuLeuGlyAla ValIleThr GlyAlaVal ValAlaAlaVal MetTrpArg* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  4601 CTCCTTGGAG CTGTGATCAC TGGAGCTGTG GTCGCTGCCG TGATGTGGAG GAGGAACCTC GACACTAGTG ACCTCGACAC CAGCGACGGC ACTACACCTC *ArgLysSer SerAspArgLys GlyGlySer TyrThrGln AlaAlaSerSer* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  4651 GAGGAAGAGC TCAGATAGAA AAGGAGGGAG TTACACTCAG GCTGCAAGCA CTCCTTCTCG AGTCTATCTT TTCCTCCCTC AATGTGAGTC CGACGTTCGT *SAspSerAla GlnGlySer AspValSerLeu ThrAlaCys LysVal*** ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Stop  4701 GTGACAGTGC CCAGGGCTCT GATGTGTCCC TCACAGCTTG TAAAGTGTGA CACTGTCACG GGTCCCGAGA CTACACAGGG AGTGTCGAAC ATTTCACACT  4751 GACAGCTGCC TTGTGTGGGA CTGAGAGGCA AGAGTTGTTC CTGCCCTTCC CTGTCGACGG AACACACCCT GACTCTCCGT TCTCAACAAG GACGGGAAGG  4801 CTTTGTGACT TGAAGAACCC TGACTTTGTT TCTGCAAAGG CACCTGCATG GAAACACTGA ACTTCTTGGG ACTGAAACAA AGACGTTTCC GTGGACGTAC  4851 TGTCTGTGTT CGTGTAGGCA TAATGTGAGG AGGTGGGGAG AGCACCCCAC ACAGACACAA GCACATCCGT ATTACACTCC TCCACCCCTC TCGTGGGGTG  4901 CCCCTGTCCA CCATGACCCT CTTCCCACGC TGACCTGTGC TCCCTCCCCA GGGGACAGGT GGTACTGGGA GAAGGGTGCG ACTGGACACG AGGGAGGGGT  HindIII                          BamHI             SalI  ~~~~~~                           ~~~~~~            ~~  4951 AAAGCTTGAT ATCCAATTCC TGCAGCCCGG GGGATCCACT TTTTCTAGGT TTTCGAACTA TAGGTTAAGG ACGTCGGGCC CCCTAGGTGA AAAAGATCCA SalI ~~~~  5001 CGACCGATCC TGAGAACTTC AGGGTGAGTT TGGGGACCCT TGATTGTTCT GCTGGCTAGG ACTCTTGAAG TCCCACTCAA ACCCCTGGGA ACTAACAAGA  5051 TTCTTTTTCG CTATTGTAAA ATTCATGTTA TATGGAGGGG GCAAAGTTTT AAGAAAAAGC GATAACATTT TAAGTACAAT ATACCTCCCC CGTTTCAAAA  5101 CAGGGTGTTG TTTAGAATGG GAAGATGTCC CTTGTATCAC CATGGACCCT GTCCCACAAC AAATCTTACC CTTCTACAGG GAACATAGTG GTACCTGGGA  5151 CATGATAATT TTGTTTCTTT CACTTTCTAC TCTGTTGACA ACCATTGTCT GTACTATTAA AACAAAGAAA GTGAAAGATG AGACAACTGT TGGTAACAGA  5201 CCTCTTATTT TCTTTTCATT TTCTGTAACT TTTTCGTTAA ACTTTAGCTT GGAGAATAAA AGAAAAGTAA AAGACATTGA AAAAGCAATT TGAAATCGAA  5251 GCATTTGTAA CGAATTTTTA AATTCACTTT TGTTTATTTG TCAGATTGTA CGTAAACATT GCTTAAAAAT TTAAGTGAAA ACAAATAAAC AGTCTAACAT  5301 AGTACTTTCT CTAATCACTT TTTTTTCAAG GCAATCAGGG TATATTATAT TCATGAAAGA GATTAGTGAA AAAAAAGTTC CGTTAGTCCC ATATAATATA  5351 TGTACTTCAG CACAGTTTTA GAGAACAATT GTTATAATTA AATGATAAGG ACATGAAGTC GTGTCAAAAT CTCTTGTTAA CAATATTAAT TTACTATTCC  5401 TAGAATATTT CTGCATATAA ATTCTGGCTG GCGTGGAAAT ATTCTTATTG ATCTTATAAA GACGTATATT TAAGACCGAC CGCACCTTTA TAAGAATAAC  5451 GTAGAAACAA CTACACCCTG GTCATCATCC TGCCTTTCTC TTTATGGTTA CATCTTTGTT GATGTGGGAC CAGTAGTAGG ACGGAAAGAG AAATACCAAT  5501 CAATGATATA CACTGTTTGA GATGAGGATA AAATACTCTG AGTCCAAACC GTTACTATAT GTGACAAACT CTACTCCTAT TTTATGAGAC TCAGGTTTGG  5551 GGGCCCCTCT GCTAACCATG TTCATGCCTT CTTCTCTTTC CTACAGCTCC CCCGGGGAGA CGATTGGTAC AAGTACGGAA GAAGAGAAAG GATGTCGAGG  5601 TGGGCAACGT GCTGGTTGTT GTGCTGTCTC ATCATTTTGG CAAAGAATTC ACCCGTTGCA CGACCAACAA CACGACAGAG TAGTAAAACC GTTTCTTAAG  5651 CTCGACCAGT GCAGGCTGCC TATCAGAAAG TGGTGGCTGG TGTGGCTAAT GAGCTGGTCA CGTCCGACGG ATAGTCTTTC ACCACCGACC ACACCGATTA  5701 GCCCTGGCCC ACAAGTATCA CTAAGCTCGC TTTCTTGCTG TCCAATTTCT CGGGACCGGG TGTTCATAGT GATTCGAGCG AAAGAACGAC AGGTTAAAGA  5751 ATTAAAGGTT CCTTTGTTCC CTAAGTCCAA CTACTAAACT GGGGGATATT TAATTTCCAA GGAAACAAGG GATTCAGGTT GATGATTTGA CCCCCTATAA  5801 ATGAAGGGCC TTGAGCATCT GGATTCTGCC TAATAAAAAA CATTTATTTT TACTTCCCGG AACTCGTAGA CCTAAGACGG ATTATTTTTT GTAAATAAAA  5851 CATTGCAATG ATGTATTTAA ATTATTTCTG AATATTTTAC TAAAAAGGGA GTAACGTTAC TACATAAATT TAATAAAGAC TTATAAAATG ATTTTTCCCT  5901 ATGTGGGAGG TCAGTGCATT TAAAACATAA AGAAATGAAG AGCTAGTTCA TACACCCTCC AGTCACGTAA ATTTTGTATT TCTTTACTTC TCGATCAAGT  5951 AACCTTGGGA AAATACACTA TATCTTAAAC TCCATGAAAG AAGGTGAGGC TTGGAACCCT TTTATGTGAT ATAGAATTTG AGGTACTTTC TTCCACTCCG  6001 TGCAAACAGC TAATGCACAT TGGCAACAGC CCCTGATGCC TATGCCTTAT ACGTTTGTCG ATTACGTGTA ACCGTTGTCG GGGACTACGG ATACGGAATA  6051 TCATCCCTCA GAAAAGGATT CAAGTAGAGG CTTGATTTGG AGGTTAAAGT AGTAGGGAGT CTTTTCCTAA GTTCATCTCC GAACTAAACC TCCAATTTCA  6101 TTTGCTATGC TGTATTTTAC ATTACTTATT GTTTTAGCTG TCCTCATGAA AAACGATACG ACATAAAATG TAATGAATAA CAAAATCGAC AGGAGTACTT  6151 TGTCTTTTCA CTACCCATTT GCTTATCCTG CATCTCTCAG CCTTGACTCC ACAGAAAAGT GATGGGTAAA CGAATAGGAC GTAGAGAGTC GGAACTGAGG  6201 ACTCAGTTCT CTTGCTTAGA GATACCACCT TTCCCCTGAA GTGTTCCTTC TGAGTCAAGA GAACGAATCT CTATGGTGGA AAGGGGACTT CACAAGGAAG  6251 CATGTTTTAC GGCGAGATGG TTTCTCCTCG CCTGGCCACT CAGCCTTAGT GTACAAAATG CCGCTCTACC AAAGAGGAGC GGACCGGTGA GTCGGAATCA  6301 TGTCTCTGTT GTCTTATAGA GGTCTACTTG AAGAAGGAAA AACAGGGGGC ACAGAGACAA CAGAATATCT CCAGATGAAC TTCTTCCTTT TTGTCCCCCG  6351 ATGGTTTGAC TGTCCTGTGA GCCCTTCTTC CCTGCCTCCC CCACTCACAG TACCAAACTG ACAGGACACT CGGGAAGAAG GGACGGAGGG GGTGAGTGTC  6401 TGACCCGGAA TCTGCAGTGC TAGTCTCCCG GAACTATCAC TCTTTCACAG ACTGGGCCTT AGACGTCACG ATCAGAGGGC CTTGATAGTG AGAAAGTGTC  6451 TCTGCTTTGG AAGGACTGGG CTTAGTATGA AAAGTTAGGA CTGAGAAGAA AGACGAAACC TTCCTGACCC GAATCATACT TTTCAATCCT GACTCTTCTT  6501 TTTGAAAGGG GGCTTTTTGT AGCTTGATAT TCACTACTGT CTTATTACCC AAACTTTCCC CCGAAAAACA TCGAACTATA AGTGATGACA GAATAATGGG  6551 TATCATAGGC CCACCCCAAA TGGAAGTCCC ATTCTTCCTC AGGATGTTTA ATAGTATCCG GGTGGGGTTT ACCTTCAGGG TAAGAAGGAG TCCTACAAAT  6601 AGATTAGCAT TCAGGAAGAG ATCAGAGGTC TGCTGGCTCC CTTATCATGT TCTAATCGTA AGTCCTTCTC TAGTCTCCAG ACGACCGAGG GAATAGTACA  6651 CCCTTATGGT GCTTCTGGCT CTGCAGTTAT TAGCATAGTG TTACCATCAA GGGAATACCA CGAAGACCGA GACGTCAATA ATCGTATCAC AATGGTAGTT  6701 CCACCTTAAC TTCATTTTTC TTATTCAATA CCTAGGTAGG TAGATGCTAG GGTGGAATTG AAGTAAAAAG AATAAGTTAT GGATCCATCC ATCTACGATC  6751 ATTCTGGAAA TAAAATATGA GTCTCAAGTG GTCCTTGTCC TCTCTCCCAG TAAGACCTTT ATTTTATACT CAGAGTTCAC CAGGAACAGG AGAGAGGGTC  6801 TCAAATTCTG AATCTAGTTG GCAAGATTCT GAAATCAAGG CATATAATCA AGTTTAAGAC TTAGATCAAC CGTTCTAAGA CTTTAGTTCC GTATATTAGT  6851 GTAATAAGTG ATGATAGAAG GGTATATAGA AGAATTTTAT TATATGAGAG CATTATTCAC TACTATCTTC CCATATATCT TCTTAAAATA ATATACTCTC  6901 GGTGAAATCC CAGCAATTTG GGAGGCTGAG GCAGGAGAAT CGCTTGATCC CCACTTTAGG GTCGTTAAAC CCTCCGACTC CGTCCTCTTA GCGAACTAGG  6951 TGGGAGGCAG AGGTTGCAGT GAGCCAAGAT TGTGCCACTG CATTCCAGCC ACCCTCCGTC TCCAACGTCA CTCGGTTCTA ACACGGTGAC GTAAGGTCGG  7001 CAGGTGACAG CATGAGACTC CGTCACAAAA AAAAAAGAAA AAAAAGGGGG GTCCACTGTC GTACTCTGAG GCAGTGTTTT TTTTTTCTTT TTTTTCCCCC  7051 GGGGGGGCGG TGGAGCCAAG ATGACCGAAT AGGAACAGCT CCAGTACTAT CCCCCCCGCC ACCTCGGTTC TACTGGCTTA TCCTTGTCGA GGTCATGATA  7101 AGCTCCCATC GTGAGTGACG CAGAAGACGG GTGATTTCTG CATTTCCAAC TCGAGGGTAG CACTCACTGC GTCTTCTGCC CACTAAAGAC GTAAAGGTTG  7151 TGAGGTACCA GGTTCATCTC ACAGGGAAGT GCCAGGCAGT GGGTGCAGGA ACTCCATGGT CCAAGTAGAG TGTCCCTTCA CGGTCCGTCA CCCACGTCCT  7201 CAGTAGGTGC AGTGCACTGT GCATGAGCCG AAGCAGGGAC GAGGCATCAC GTCATCCACG TCACGTGACA CGTACTCGGC TTCGTCCCTG CTCCGTAGTG  7251 CTCACCCGGG AAGCACAAGG GGTCAGGGAA TTCCCTTTCC TAGTCAAAGA GAGTGGGCCC TTCGTGTTCC CCAGTCCCTT AAGGGAAAGG ATCAGTTTCT  7301 AAAGGGTGAC AGATGGCACC TGGAAAATCG GGTCACTCCC GCCCTAATAC TTTCCCACTG TCTACCGTGG ACCTTTTAGC CCAGTGAGGG CGGGATTATG                HindIII                ~~~~~~~  7351 TGCGCTCTTC CAACAAGCTT GTCTTTGGAA AATAGATCAA TTTCCCTTGG ACGCGAGAAG GTTGTTCGAA CAGAAACCTT TTATCTAGTT AAAGGGAACC  7401 GAAGAAGATT TTTAGCACAG CAAGGGGCAG GATGTTCAAC TGTGAGAAAA CTTCTTCTAA AAATCGTGTC GTTCCCCGTC CTACAAGTTG ACACTCTTTT  7451 CGAAGAATTA GCCAAAAAAC TTCCAGTAAG CCTGCAAAAA AAAAAAAAAA GCTTCTTAAT CGGTTTTTTG AAGGTCATTC GGACGTTTTT TTTTTTTTTT  7501 ATAAAAGCTA AGTTTCTATA AATGTTCTGT AAATGTAAAA CAGAAGGTAA TATTTTCGAT TCAAAGATAT TTACAAGACA TTTACATTTT GTCTTCCATT  7551 GTCAACTGCA CCTAATAAAA ATCACTTAAT AGCAATGTGC TGTGTCAGTT CAGTTGACGT GGATTATTTT TAGTGAATTA TCGTTACACG ACACAGTCAA  7601 GTTTATTGGA ACCACACCCG GTACACATCC TGTCCAGCAT TTGCAGTGCG CAAATAACCT TGGTGTGGGC CATGTGTAGG ACAGGTCGTA AACGTCACGC  7651 TGCATTGAAT TATTGTGCTG GCTAGACTTC ATGGCGCCTG GCACCGAATC ACGTAACTTA ATAACACGAC CGATCTGAAG TACCGCGGAC CGTGGCTTAG  7701 CTGCCTTCTC AGCGAAAATG AATAATTGCT TTGTTGGCAA GAAACTAAGC GACGGAAGAG TCGCTTTTAC TTATTAACGA AACAACCGTT CTTTGATTCG  7751 ATCAATGGGA CGCGTGCAAA GCACCGGCGG CGGTAGATGC GGGGTAAGTA TAGTTACCCT GCGCACGTTT CGTGGCCGCC GCCATCTACG CCCCATTCAT  7801 CTGAATTTTA ATTCGACCTA TCCCGGTAAA GCGAAAGCGA CACGCTTTTT GACTTAAAAT TAAGCTGGAT AGGGCCATTT CGCTTTCGCT GTGCGAAAAA  7851 TTTCACACAT AGCGGGACCG AACACGTTAT AAGTATCGAT TAGGTCTATT AAAGTGTGTA TCGCCCTGGC TTGTGCAATA TTCATAGCTA ATCCAGATAA  7901 TTTGTCTCTC TGTCGGAACC AGAACTGGTA AAAGTTTCCA TTGCGTCTGG AAACAGAGAG ACAGCCTTGG TCTTGACCAT TTTCAAAGGT AACGCAGACC  7951 GCTTGTCTAT CATTGCGTCT CTATGGTTTT TGGAGGATTA GACGGGGCCA CGAACAGATA GTAACGCAGA GATACCAAAA ACCTCCTAAT CTGCCCCGGT  8001 CCAGTAATGG TGCATAGCGG ATGTCTGTAC CGCCATCGGT GCACCGATAT GGTCATTACC ACGTATCGCC TACAGACATG GCGGTAGCCA CGTGGCTATA  8051 AGGTTTGGGG CTCCCCAAGG GACTGCTGGG ATGACAGCTT CATATTATAT TCCAAACCCC GAGGGGTTCC CTGACGACCC TACTGTCGAA GTATAATATA  8101 TGAATGGGCG CATAATCAGC TTAATTGGTG AGGACAAGCT ACAAGTTGTA ACTTACCCGC GTATTAGTCG AATTAACCAC TCCTGTTCGA TGTTCAACAT  8151 ACCTGATCTC CACAAAGTAC GTTGCCGGTC GGGGTCAAAC CGTCTTCGGT TGGACTAGAG GTGTTTCATG CAACGGCCAG CCCCAGTTTG GCAGAAGCCA  8201 GCTCGAAACC GCCTTAAACT ACAGACAGGT CCCAGCCAAG TAGGCGGATC CGAGCTTTGG CGGAATTTGA TGTCTGTCCA GGGTCGGTTC ATCCGCCTAG  8251 AAAACCTCAA AAAGGCGGGA GCCAATCAAA ATGCAGCATT ATATTTTAAG TTTTGGAGTT TTTCCGCCCT CGGTTAGTTT TACGTCGTAA TATAAAATTC  8301 CTCACCGAAA CCGGTAAGTA AAGACTATGT ATTTTTTCCC AGTGAATAAT GAGTGGCTTT GGCCATTCAT TTCTGATACA TAAAAAAGGG TCACTTATTA  Start E1 SEQ ID NO: 4   MetAlaAsn AspLysGly SerAsnTrp                          ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8351 TGTTGTTAAC TATAAAAAGC GTCATGGCAA ACGATAAAGG TAGCAATTGG ACAACAATTG ATATTTTTCG CAGTACCGTT TGCTATTTCC ATCGTTAACC AspSerGlyLeu GlyCysSer TyrLeuLeu ThrGluAlaGlu CysGluSer* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8401 GATTCGGGCT TGGGATGCTC ATATCTGCTG ACTGAGGCAG AATGTGAAAG CTAAGCCCGA ACCCTACGAG TATAGACGAC TGACTCCGTC TTACACTTTC *AspLysGlu AsnGluGluPro GlyAlaGly ValGluLeu SerValGluSer* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8451 TGACAAAGAG AATGAGGAAC CCGGGGCAGG TGTAGAACTG TCTGTGGAAT ACTGTTTCTC TTACTCCTTG GGCCCCGTCC ACATCTTGAC AGACACCTTA *SAspArgTyr AspSerGln AspGluAspPhe ValAspAsn AlaSerVal ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8501 CTGATCGGTA TGATAGCCAG GATGAGGATT TTGTTGACAA TGCATCAGTC GACTAGCCAT ACTATCGGTC CTACTCCTAA AACAACTGTT ACGTAGTCAG PheGlnGlyAsn HisLeuGlu ValPheGln AlaLeuGluLys LysAlaGly* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8551 TTTCAGGGAA ATCACCTGGA GGTCTTCCAG GCATTAGAGA AAAAGGCGGG AAAGTCCCTT TAGTGGACCT CCAGAAGGTC CGTAATCTCT TTTTCCGCCC *GluGluGln IleLeuAsnLeu LysArgLys ValLeuGly SerSerGlnAsn* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8601 TGAGGAGCAG ATTTTAAATT TGAAAAGAAA AGTATTGGGG AGTTCGCAAA ACTCCTCGTC TAAAATTTAA ACTTTTCTTT TCATAACCCC TCAAGCGTTT *ASerSerGly SerGluAla SerGluThrPro ValLysArg ArgLysSer ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8651 ACAGCAGCGG TTCCGAAGCA TCTGAAACTC CAGTTAAAAG ACGGAAATCA TGTCGTCGCC AAGGCTTCGT AGACTTTGAG GTCAATTTTC TGCCTTTAGT GlyAlaLysArg ArgLeuPhe AlaGluAsn GluAlaAsnArg ValLeuThr* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8701 GGAGCAAAGC GAAGATTATT TGCTGAAAAT GAAGCTAACC GTGTTCTTAC CCTCGTTTCG CTTCTAATAA ACGACTTTTA CTTCGATTGG CACAAGAATG *ProLeuGln ValGlnGlyGlu GlyGluGly ArgGlnGlu LeuAsnGluGlu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8751 GCCCCTCCAG GTACAGGGGG AGGGGGAGGG GAGGCAAGAA CTTAATGAGG CGGGGAGGTC CATGTCCCCC TCCCCCTCCC CTCCGTTCTT GAATTACTCC *GGlnAlaIle SerHisLeu HisLeuGlnLeu ValLysSer LysAsnAla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8801 AGCAGGCAAT TAGTCATCTA CATCTGCAGC TTGTTAAATC TAAAAATGCT TCGTCCGTTA ATCAGTAGAT GTAGACGTCG AACAATTTAG ATTTTTACGA ThrValPheLys LeuGlyLeu PheLysSer LeuPheLeuCys SerPheHis* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8851 ACAGTTTTTA AGCTGGGGCT CTTTAAATCT TTGTTCCTTT GTAGCTTCCA TGTCAAAAAT TCGACCCCGA GAAATTTAGA AACAAGGAAA CATCGAAGGT *AspIleThr ArgLeuPheLys AsnAspLys ThrThrAsn GlnGlnTrpVal* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8901 TGATATTACG AGGTTGTTTA AGAATGATAA GACCACTAAT CAGCAATGGG ACTATAATGC TCCAACAAAT TCTTACTATT CTGGTGATTA GTCGTTACCC *VLeuAlaVal PheGlyLeu AlaGluValPhe PheGluAla SerPheGlu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8951 TGCTGGCTGT GTTTGGCCTT GCAGAGGTGT TTTTTGAGGC GAGTTTCGAA ACGACCGACA CAAACCGGAA CGTCTCCACA AAAAACTCCG CTCAAAGCTT LeuLeuLysLys GlnCysSer PheLeuGln MetGlnLysArg SerHisGlu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9001 CTCCTAAAGA AGCAGTGTAG TTTTCTGCAG ATGCAAAAAA GATCTCATGA GAGGATTTCT TCGTCACATC AAAAGACGTC TACGTTTTTT CTAGAGTACT *GlyGlyThr CysAlaValTyr LeuIleCys PheAsnThr AlaLysSerArg* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9051 AGGAGGAACT TGTGCAGTTT ACTTAATCTG CTTTAACACA GCTAAAAGCA TCCTCCTTGA ACACGTCAAA TGAATTAGAC GAAATTGTGT CGATTTTCGT *AGluThrVal ArgAsnLeu MetAlaAsnMet LeuAsnVal ArgGluGlu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9101 GAGAAACAGT CCGGAATCTG ATGGCAAACA TGCTAAATGT AAGAGAAGAG CTCTTTGTCA GGCCTTAGAC TACCGTTTGT ACGATTTACA TTCTCTTCTC CysLeuMetLeu GlnProPro LysIleArg GlyLeuSerAla AlaLeuPhe* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9151 TGTTTGATGC TGCAGCCACC TAAAATTCGA GGACTCAGCG CAGCTCTATT ACAAACTACG ACGTCGGTGG ATTTTAAGCT CCTGAGTCGC GTCGAGATAA *TrpPheLys SerSerLeuSer ProAlaThr LeuLysHis GlyAlaLeuPro* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9201 CTGGTTTAAA AGTAGTTTGT CACCCGCTAC ACTTAAACAT GGTGCTTTAC GACCAAATTT TCATCAAACA GTGGGCGATG TGAATTTGTA CCACGAAATG *PGluTrpIle ArgAlaGln ThrThrLeuAsn GluSerLeu GlnThrGlu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9251 CTGAGTGGAT ACGGGCGCAA ACTACTCTGA ACGAGAGCTT GCAGACCGAG GACTCACCTA TGCCCGCGTT TGATGAGACT TGCTCTCGAA CGTCTGGCTC LysPheAspPhe GlyThrMet ValGlnTrp AlaTyrAspHis LysTyrAla* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9301 AAATTCGACT TCGGAACTAT GGTGCAATGG GCCTATGATC ACAAATATGC TTTAAGCTGA AGCCTTGATA CCACGTTACC CGGATACTAG TGTTTATACG *GluGluSer LysIleAlaTyr GluTyrAla LeuAlaAla GlySerAspSer* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9351 TGAGGAGTCT AAAATAGCCT ATGAATATGC TTTGGCTGCA GGATCTGATA ACTCCTCAGA TTTTATCGGA TACTTATACG AAACCGACGT CCTAGACTAT *SAsnAlaArg AlaPheLeu AlaThrAsnSer GlnAlaLys HisValLys ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9401 GCAATGCACG GGCTTTTTTA GCAACTAACA GCCAAGCTAA GCATGTGAAG CGTTACGTGC CCGAAAAAAT CGTTGATTGT CGGTTCGATT CGTACACTTC AspCysAlaThr MetValArg HisTyrLeu ArgAlaGluThr GlnAlaLeu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9451 GACTGTGCAA CTATGGTAAG ACACTATCTA AGAGCTGAAA CACAAGCATT CTGACACGTT GATACCATTC TGTGATAGAT TCTCGACTTT GTGTTCGTAA *SerMetPro AlaTyrIleLys AlaArgCys LysLeuAla ThrGlyGluGly* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9501 AAGCATGCCT GCATATATTA AAGCTAGGTG CAAGCTGGCA ACTGGGGAAG TTCGTACGGA CGTATATAAT TTCGATCCAC GTTCGACCGT TGACCCCTTC *GSerTrpLys SerIleLeu ThrPhePheAsn TyrGlnAsn IleGluLeu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9551 GAAGCTGGAA GTCTATCCTA ACTTTTTTTA ACTATCAGAA TATTGAATTA CTTCGACCTT CAGATAGGAT TGAAAAAAAT TGATAGTCTT ATAACTTAAT IleThrPheIle AsnAlaLeu LysLeuTrp LeuLysGlyIle ProLysLys* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9601 ATTACCTTTA TTAATGCTTT AAAGCTCTGG CTAAAAGGAA TTCCAAAAAA TAATGGAAAT AATTACGAAA TTTCGAGACC GATTTTCCTT AAGGTTTTTT *AsnCysLeu AlaPheIleGly ProProAsn ThrGlyLys SerMetLeuCys* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9651 AAACTGTTTA GCATTTATTG GCCCTCCAAA CACAGGCAAG TCTATGCTCT TTTGACAAAT CGTAAATAAC CGGGAGGTTT GTGTCCGTTC AGATACGAGA *CAsnSerLeu IleHisPhe LeuGlyGlySer ValLeuSer PheAlaAsn ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9701 GCAACTCATT AATTCATTTT TTGGGTGGTA GTGTTTTATC TTTTGCCAAC CGTTGAGTAA TTAAGTAAAA AACCCACCAT CACAAAATAG AAAACGGTTG HisLysSerHis PheTrpLeu AlaSerLeu AlaAspThrArg AlaAlaLeu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9751 CATAAAAGTC ACTTTTGGCT TGCTTCCCTA GCAGATACTA GAGCTGCTTT GTATTTTCAG TGAAAACCGA ACGAAGGGAT CGTCTATGAT CTCGACGAAA *ValAspAsp AlaThrHisAla CysTrpArg TyrPheAsp ThrTyrLeuArg* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9801 AGTAGATGAT GCTACTCATG CTTGCTGGAG GTACTTTGAC ACATACCTCA TCATCTACTA CGATGAGTAC GAACGACCTC CATGAAACTG TGTATGGAGT *AAsnAlaLeu AspGlyTyr ProValSerIle AspArgLys HisLysAla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9851 GAAATGCATT GGATGGCTAC CCTGTCAGTA TTGATAGAAA ACACAAAGCA CTTTACGTAA CCTACCGATG GGACAGTCAT AACTATCTTT TGTGTTTCGT AlaValGlnIle LysAlaPro ProLeuLeu ValThrSerAsn IleAspVal* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9901 GCGGTTCAAA TTAAAGCTCC ACCCCTCCTG GTAACCAGTA ATATTGATGT CGCCAAGTTT AATTTCGAGG TGGGGAGGAC CATTGGTCAT TATAACTACA *GlnAlaGlu AspArgTyrLeu TyrLeuHis SerArgVal GlnThrPheArg* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9951 GCAGGCAGAG GACAGATATT TGTACTTGCA TAGTCGGGTG CAAACCTTTC CGTCCGTCTC CTGTCTATAA ACATGAACGT ATCAGCCCAC GTTTGGAAAG *APheGluGln ProCysThr AspGluSerGly GluGlnPro PheAsnIle ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10001 GCTTTGAGCA GCCATGCACA GATGAATCGG GTGAGCAACC TTTTAATATT CGAAACTCGT CGGTACGTGT CTACTTAGCC CACTCGTTGG AAAATTATAA ThrAspAlaAsp TrpLysSer PhePheVal ArgLeuTrpGly ArgLeuAsp* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10051 ACTGATGCAG ATTGGAAATC TTTTTTTGTA AGGTTATGGG GGCGTTTAGA TGACTACGTC TAACCTTTAG AAAAAAACAT TCCAATACCC CCGCAAATCT              SEQ ID NO: 5 Start E2 MetGluThr AlaCysGlu                                    ~~~~~~~~~~~~~~~~~~~ *LeuIleAsp GluGluGluAsp SerGluGlu AspGlyAsp SerMetArgThr* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10101 CCTGATTGAC GAGGAGGAGG ATAGTGAAGA GGATGGAGAC AGCATGCGAA GGACTAACTG CTCCTCCTCC TATCACTTCT CCTACCTCTG TCGTACGCTT ArgLeuHisAla AlaGlnGlu ThrGlnMet GlnLeuIleGlu LysSerSer* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *TPheThrCys SerAlaArg AsnThrAsnAla ValAsp Stop E1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10151 CGTTTACATG CAGCGCAAGA AACACAAATG CAGTTGATTG AGAAAAGTAG GCAAATGTAC GTCGCGTTCT TTGTGTTTAC GTCAACTAAC TCTTTTCATC *AspLysLeu GlnAspHisIle LeuTyrTrp ThrAlaVal ArgThrGluAsn* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10201 TGATAAGTTG CAAGATCATA TACTGTACTG GACTGCTGTT AGAACTGAGA ACTATTCAAC GTTCTAGTAT ATGACATGAC CTGACGACAA TCTTGACTCT *AThrLeuLeu TyrAlaAla ArgLysLysGly ValThrVal LeuGlyHis ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10251 ACACACTGCT TTATGCTGCA AGGAAAAAAG GGGTGACTGT CCTAGGACAC TGTGTGACGA AATACGACGT TCCTTTTTTC CCCACTGACA GGATCCTGTG CysArgValPro HisSerVal ValCysGln GluArgAlaLys GlnAlaIle* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10301 TGCAGAGTAC CACACTCTGT AGTTTGTCAA GAGAGAGCCA AGCAGGCCAT ACGTCTCATG GTGTGAGACA TCAAACAGTT CTCTCTCGGT TCGTCCGGTA *GluMetGln LeuSerLeuGln GluLeuSer LysThrGlu PheGlyAspGlu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10351 TGAAATGCAG TTGTCTTTGC AGGAGTTAAG CAAAACTGAG TTTGGGGATG ACTTTACGTC AACAGAAACG TCCTCAATTC GTTTTGACTC AAACCCCTAC *GProTrpSer LeuLeuAsp ThrSerTrpAsp ArgTyrMet SerGluPro ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10401 AACCATGGTC TTTGCTTGAC ACAAGCTGGG ACCGATATAT GTCAGAACCT TTGGTACCAG AAACGAACTG TGTTCGACCC TGGCTATATA CAGTCTTGGA LysArgCysPhe LysLysGly AlaArgVal ValGluValGlu PheAspGly* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10451 AAACGGTGCT TTAAGAAAGG CGCCAGGGTG GTAGAGGTGG AGTTTGATGG TTTGCCACGA AATTCTTTCC GCGGTCCCAC CATCTCCACC TCAAACTACC *AsnAlaSer AsnThrAsnTrp TyrThrVal TyrSerAsn LeuTyrMetArg* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10501 AAATGCAAGC AATACAAACT GGTACACTGT CTACAGCAAT TTGTACATGC TTTACGTTCG TTATGTTTGA CCATGTGACA GATGTCGTTA AACATGTACG *AThrGluAsp GlyTrpGln LeuAlaLysAla GlyLeuThr GluLeuGly ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10551 GCACAGAGGA CGGCTGGCAG CTTGCGAAGG CTGGGCTGAC GGAACTGGGC CGTGTCTCCT GCCGACCGTC GAACGCTTCC GACCCGACTG CCTTGACCCG SerThrThrAla ProTrpPro ValLeuAsp AlaPheThrIle LeuAlaLeu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10601 TCTACTACTG CACCATGGCC GGTGCTGGAC GCATTTACTA TTCTCGCTTT AGATGATGAC GTGGTACCGG CCACGACCTG CGTAAATGAT AAGAGCGAAA *ValThrArg GlnProAspLeu ValGlnGln GlyIleThr Leu Stop E2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10651 GGTGACGAGG CAGCCAGATT TAGTACAACA GGGCATTACT CTGTAAGAGA CCACTGCTCC GTCGGTCTAA ATCATGTTGT CCCGTAATGA GACATTCTCT 10701 TCAGGACAGA GTGTATGCTG GTGTCTCATC CACCTCTTCT GATTTTAGAG AGTCCTGTCT CACATACGAC CACAGAGTAG GTGGAGAAGA CTAAAATCTC 10751 ATCGCCCAGA CGGAGTCTGG GTCGCATCCG AAGGACCTGA AGGAGACCCT TAGCGGGTCT GCCTCAGACC CAGCGTAGGC TTCCTGGACT TCCTCTGGGA 10801 GCAGGAAAAG AAGCCGAGCC AGCCCAGCCT GTCTCTTCTT TGCTCGGCTC CGTCCTTTTC TTCGGCTCGG TCGGGTCGGA CAGAGAAGAA ACGAGCCGAG 10851 CCCCGCCTGC GGTCCCATCA GAGCAGGCCT CGGTTGGGTA CGGGACGGTC GGGGCGGACG CCAGGGTAGT CTCGTCCGGA GCCAACCCAT GCCCTGCCAG 10901 CTCGCTCGCA CCCCTACAAT TTTCCTGCAG GCTCGGGGGG CTCTATTCTC GAGCGAGCGT GGGGATGTTA AAAGGACGTC CGAGCCCCCC GAGATAAGAG 10951 CGCTCTTCCT CCACCCCGTG CAGGGCACGG TACCGGTGGA CTTGGCATCA GCGAGAAGGA GGTGGGGCAC GTCCCGTGCC ATGGCCACCT GAACCGTAGT 11001 AGGCAGGAAG AAGAGGAGCA GTCGCCCGAC TCCACAGAGG AAGAACCAGT TCCGTCCTTC TTCTCCTCGT CAGCGGGCTG AGGTGTCTCC TTCTTGGTCA 11051 GACTCTCCCA AGGCGCACCA CCAATGATGG ATTCCACCTG TTAAAGGCAG CTGAGAGGGT TCCGCGTGGT GGTTACTACC TAAGGTGGAC AATTTCCGTC 11101 GAGGGTCATG CTTTGCTCTA ATTTCAGGAA CTGCTAACCA GGTAAAGTGC CTCCCAGTAC GAAACGAGAT TAAAGTCCTT GACGATTGGT CCATTTCACG 11151 TATCGCTTTC GGGTGAAAAA GAACCATAGA CATCGCTACG AGAACTGCAC ATAGCGAAAG CCCACTTTTT CTTGGTATCT GTAGCGATGC TCTTGACGTG 11201 CACCACCTGG TTCACAGTTG CTGACAACGG TGCTGAAAGA CAAGGACAAG GTGGTGGACC AAGTGTCAAC GACTGTTGCC ACGACTTTCT GTTCCTGTTC 11251 CACAAATACT GATCACCTTT GGATCGCCAA GTCAAAGGCA AGACTTTCTG GTGTTTATGA CTAGTGGAAA CCTAGCGGTT CAGTTTCCGT TCTGAAAGAC 11301 AAACATGTAC CACTACCTCC TGGAATGAAC ATTTCCGGCT TTACAGCCAG TTTGTACATG GTGATGGAGG ACCTTACTTG TAAAGGCCGA AATGTCGGTC 11351 CTTGGACTTC TGATCACTGC CATTGCCTTT TCTTCATCTG ACTGGTGTAC GAACCTGAAG ACTAGTGACG GTAACGGAAA AGAAGTAGAC TGACCACATG 11401 TATGCCAAAT CTATGCGACC GCATTATAAA GCCGAATTCT GCAGATATCC ATACGGTTTA GATACGCTGG CGTAATATTT CGGCTTAAGA CGTCTATAGG 11451 ATCACACTGG CGGCCATATG GCCGCTATGC GGTGTGAAAT ACCGCACAGA TAGTGTGACC GCCGGTATAC CGGCGATACG CCACACTTTA TGGCGTGTCT 11501 TGCGTAAGGA GAAAATACCG CATCAGGCGC TCTTCCGCTT CCTCGCTCAC ACGCATTCCT CTTTTATGGC GTAGTCCGCG AGAAGGCGAA GGAGCGAGTG 11551 TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT ACTGAGCGAC GCGAGCCAGC AAGCCGACGC CGCTCGCCAT AGTCGAGTGA 11601 CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG GTTTCCGCCA TTATGCCAAT AGGTGTCTTA GTCCCCTATT GCGTCCTTTC 11651 AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC TTGTACACTC GTTTTCCGGT CGTTTTCCGG TCCTTGGCAT TTTTCCGGCG 11701 GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA CAACGACCGC AAAAAGGTAT CCGAGGCGGG GGGACTGCTC GTAGTGTTTT 11751 ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC TAGCTGCGAG TTCAGTCTCC ACCGCTTTGG GCTGTCCTGA TATTTCTATG 11801 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GTCCGCAAAG GGGGACCTTC GAGGGAGCAC GCGAGAGGAC AAGGCTGGGA 11851 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC CGGCGAATGG CCTATGGACA GGCGGAAAGA GGGAAGCCCT TCGCACCGCG 11901 TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC AAAGAGTATC GAGTGCGACA TCCATAGAGT CAAGCCACAT CCAGCAAGCG 11951 TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC AGGTTCGACC CGACACACGT GCTTGGGGGG CAAGTCGGGC TGGCGACGCG 12001 CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT GAATAGGCCA TTGATAGCAG AACTCAGGTT GGGCCATTCT GTGCTGAATA 12051 CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA GCGGTGACCG TCGTCGGTGA CCATTGTCCT AATCGTCTCG CTCCATACAT 12101 GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG CCGCCACGAT GTCTCAAGAA CTTCACCACC GGATTGATGC CGATGTGATC 12151 AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA TTCCTGTCAT AAACCATAGA CGCGAGACGA CTTCGGTCAA TGGAAGCCTT 12201 AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT TTTCTCAACC ATCGAGAACT AGGCCGTTTG TTTGGTGGCG ACCATCGCCA 12251 GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA CCAAAAAAAC AAACGTTCGT CGTCTAATGC GCGTCTTTTT TTCCTAGAGT 12301 AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA TCTTCTAGGA AACTAGAAAA GATGCCCCAG ACTGCGAGTC ACCTTGCTTT 12351 ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC TGAGTGCAAT TCCCTAAAAC CAGTACTCTA ATAGTTTTTC CTAGAAGTGG 12401 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA ATCTAGGAAA ATTTAATTTT TACTTCAAAA TTTAGTTAGA TTTCATATAT 12451 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA ACTCATTTGA ACCAGACTGT CAATGGTTAC GAATTAGTCA CTCCGTGGAT       Stop SEQ ID NO: 6  TrpHis LysIleLeuSer AlaGlyIle*                          ***~~~~~~~~~~~~~~~~~~~~~~~~~~ 12501 TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC AGAGTCGCTA GACAGATAAA GCAAGTAGGT ATCAACGGAC TGAGGGGCAG _(*)GluAlaIle GlnArgAsnArg GluAspMet ThrAlaGln SerGlyThrThr* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12551 GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC CACATCTATT GATGCTATGC CCTCCCGAAT GGTAGACCGG GGTCACGACG _(*) _(*)TyrIleVal ValIleArg SerProLysGly AspProGly LeuAlaAla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12601 AATGATACCG CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA TTACTATGGC GCTCTGGGTG CGAGTGGCCG AGGTCTAAAT AGTCGTTATT IleIleGlyArg SerGlyArg GluGlyAla GlySerLysAsp AlaIlePhe* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12651 ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC TGGTCGGTCG GCCTTCCCGG CTCGCGTCTT CACCAGGACG TTGAAATAGG _(*)TrpGlyAla ProLeuAlaSer ArgLeuLeu ProGlyAla ValLysAspAla* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12701 GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC CGGAGGTAGG TCAGATAATT AACAACGGCC CTTCGATCTC ATTCATCAAG _(*) _(*)GluMetTrp AspIleLeu GlnGlnArgSer AlaLeuThr LeuLeuGlu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12751 GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTGCA GGCATCGTGG CGGTCAATTA TCAAACGCGT TGCAACAACG GTAACGACGT CCGTAGCACC GlyThrLeuLeu LysArgLeu ThrThrAla MetAlaAlaPro MetThrThr* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12801 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA ACAGTGCGAG CAGCAAACCA TACCGAAGTA AGTCGAGGCC AAGGGTTGCT _(*)AspArgGlu AspAsnProIle AlaGluAsn LeuGluPro GluTrpArgAsp* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12851 TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC AGTTCCGCTC AATGTACTAG GGGGTACAAC ACGTTTTTTC GCCAATCGAG _(*) _(*)LeuArgThr ValHisAsp GlyMetAsnHis LeuPheAla ThrLeuGlu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12901 CTTCGGTCCT CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC GAAGCCAGGA GGCTAGCAAC AGTCTTCATT CAACCGGCGT CACAATAGTG LysProGlyGly IleThrThr LeuLeuLeu AsnAlaAlaThr AsnAspSer* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12951 TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA AGTACCAATA CCGTCGTGAC GTATTAAGAG AATGACAGTA CGGTAGGCAT _(*)MetThrIle AlaAlaSerCys LeuGluArg ValThrMet GlyAspThrLeu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13001 AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA TCTACGAAAA GACACTGACC ACTCATGAGT TGGTTCAGTA AGACTCTTAT _(*) _(*)HisLysGlu ThrValPro SerTyrGluVal LeuAspAsn GlnSerTyr ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13051 GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAACA CGGGATAATA CACATACGCC GCTGGCTCAA CGAGAACGGG CCGCAGTTGT GCCCTATTAT HisIleArgArg GlyLeuGln GluGlnGly AlaAspValArg SerLeuVal* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13101 CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT GGCGCGGTGT ATCGTCTTGA AATTTTCACG AGTAGTAACC TTTTGCAAGA _(*)AlaGlyCys LeuLeuValLys PheThrSer MetMetPro PheArgGluGlu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13151 TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT AGCCCCGCTT TTGAGAGTTC CTAGAATGGC GACAACTCTA GGTCAAGCTA _(*) _(*)ProArgPhe SerGluLeu IleLysGlySer AsnLeuAsp LeuGluIle ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13201 GTAACCCACT CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA CATTGGGTGA GCACGTGGGT TGACTAGAAG TCGTAGAAAA TGAAAGTGGT TyrGlyValArg AlaGlyLeu GlnAspGlu AlaAspLysVal LysValLeu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13251 GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA CGCAAAGACC CACTCGTTTT TGTCCTTCCG TTTTACGGCG TTTTTTCCCT _(*)ThrGluPro HisAlaPheVal ProLeuCys PheAlaAla PhePheProIle* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13301 ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TATTCCCGCT GTGCCTTTAC AACTTATGAG TATGAGAAGG AAAAAGTTAT _(*) _(*) LeuAlaVal ArgPheHis GlnIleSerMet Start Beta-Lactamase ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13351 TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AATAACTTCG TAAATAGTCC CAATAACAGA GTACTCGCCT ATGTATAAAC 13401 AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA TTACATAAAT CTTTTTATTT GTTTATCCCC AAGGCGCGTG TAAAGGGGCT 13451 AAAGTGCCAC CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA TTTCACGGTG GACTGCAGAT TCTTTGGTAA TAATAGTACT GTAATTGGAT 13501 TAAAAATAGG CGTATCACGA GGCCCTTTCG TCTTCAAGAA TTCTCATGTT ATTTTTATCC GCATAGTGCT CCGGGAAAGC AGAAGTTCTT AAGAGTACAA                    HindIII                    ~~~~~~~ 13551 TGACAGCTTA TCATCGATAA GCTTCACGCT GCCGCAAGCA CTCAGGGCGC ACTGTCGAAT AGTAGCTATT CGAAGTGCGA CGGCGTTCGT GAGTCCCGCG 13601 AAGGGCTGCT AAAGGAAGCG GAACACGTAG AAAGCCAGTC CGCAGAAACG TTCCCGACGA TTTCCTTCGC CTTGTGCATC TTTCGGTCAG GCGTCTTTGC 13651 GTGCTGACCC CGGATGAATG TCAGCTACTG GGCTATCTGG ACAAGGGAAA CACGACTGGG GCCTACTTAC AGTCGATGAC CCGATAGACC TGTTCCCTTT 13701 ACGCAAGCGC AAAGAGAAAG CAGGTAGCTT GCAGTGGGCT TACATGGCGA TGCGTTCGCG TTTCTCTTTC GTCCATCGAA CGTCACCCGA ATGTACCGCT 13751 TAGCTAGACT GGGCGGTTTT ATGGACAGCA AGCGAACCGG AATTGCCAGC ATCGATCTGA CCCGCCAAAA TACCTGTCGT TCGCTTGGCC TTAACGGTCG 13801 TGGGGCGCCC TCTGGTAAGG TTGGGAAGCC CTGCAAAGTA AACTGGATGG ACCCCGCGGG AGACCATTCC AACCCTTCGG GACGTTTCAT TTGACCTACC 13851 CTTTCTTGCC GCCAAGGATC TGATGGCGCA GGGGATCAAG ATCCTGCTTC GAAAGAACGG CGGTTCCTAG ACTACCGCGT CCCCTAGTTC TAGGACGAAG 13901 ATCCCCGTGG CCCGTTGCTC GCGTTTGCTG GCGGTGTCCC CGGAAGAAAT TAGGGGCACC GGGCAACGAG CGCAAACGAC CGCCACAGGG GCCTTCTTTA 13951 ATATTTGCAT GTCTTTAGTT CTATGATGAC ACAAACCCCG CCCAGCGTCT TATAAACGTA CAGAAATCAA GATACTACTG TGTTTGGGGC GGGTCGCAGA 14001 TGTCATTGGC GAATTCGAAC ACGCAGATGC AGTCGGGGCG GCGCGGTCCC ACAGTAACCG CTTAAGCTTG TGCGTCTACG TCAGCCCCGC CGCGCCAGGG 14051 AGGTCCACTT CGCATATTAA GGTGACGCGT GTGGCCTCGA ACACCGAGCG TCCAGGTGAA GCGTATAATT CCACTGCGCA CACCGGAGCT TGTGGCTCGC 14101 ACCCTGCAGC GACCCGCTTA ACAGCGTCAA CAGCGTGCCG CAGATCTGAT TGGGACGTCG CTGGGCGAAT TGTCGCAGTT GTCGCACGGC GTCTAGACTA Start  G418 resist SEQ ID NO: 7      Met IleGluGlnAsp GlyLeuHis*                                      ~~~~~~~~~~~~~~~~~~~~~~~~~ 14151 CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG ATGGATTGCA GTTCTCTGTC CTACTCCTAG CAAAGCGTAC TAACTTGTTC TACCTAACGT *AlaGlySer ProAlaAlaTrp ValGluArg LeuPheGly TyrAspTrpAla* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14201 CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG GCGTCCAAGA GGCCGGCGAA CCCACCTCTC CGATAAGCCG ATACTGACCC *AGlnGlnThr IleGlyCys SerAspAlaAla ValPheArg LeuSerAla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14251 CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG GTGTTGTCTG TTAGCCGACG AGACTACGGC GGCACAAGGC CGACAGTCGC GlnGlyArgPro ValLeuPhe ValLysThr AspLeuSerGly AlaLeuAsn* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14301 CAGGGGCGCC CGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA GTCCCCGCGG GCCAAGAAAA ACAGTTCTGG CTGGACAGGC CACGGGACTT *GluLeuGln AspGluAlaAla ArgLeuSer TrpLeuAla ThrThrGlyVal* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14351 TGAACTGCAG GACGAGGCAG CGCGGCTATC GTGGCTGGCC ACGACGGGCG ACTTGACGTC CTGCTCCGTC GCGCCGATAG CACCGACCGG TGCTGCCCGC *VProCysAla AlaValLeu AspValValThr GluAlaGly ArgAspTrp ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14401 TTCCTTGCGC AGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG AAGGAACGCG TCGACACGAG CTGCAACAGT GACTTCGCCC TTCCCTGACC LeuLeuLeuGly GluValPro GlyGlnAsp LeuLeuSerSer HisLeuAla* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14451 CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC GACGATAACC CGCTTCACGG CCCCGTCCTA GAGGACACTA GAGTGGAACG                                           ~~~~~~~~ *ProAlaGlu LysValSerIle MetAlaAsp AlaMetArg ArgLeuHisThr* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14501 TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA AGGACGGCTC TTTCATAGGT ACTACCGACT ACGTTACGCC GCCGACGTAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *TLeuAspPro AlaThrCys ProPheAspHis GlnAlaLys HisArgIle ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14551 CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GCGAACTAGG CCGATGGACG GGTAAGCTGG TGGTTCGCTT TGTAGCGTAG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ GluArgAlaArg ThrArgMet GluAlaGly LeuValAspGln AspAspLeu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14601 GAGCGAGCAC GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT CTCGCTCGTG CATGAGCCTA CCTTCGGCCA GAACAGCTAG TCCTACTAGA ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *AspGluGlu HisGlnGlyLeu AlaProAla GluLeuPhe AlaArgLeuLys* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14651 GGACGAAGAG CATCAGGGGC TCGCGCCAGC CGAACTGTTC GCCAGGCTCA CCTGCTTCTC GTAGTCCCCG AGCGCGGTCG GCTTGACAAG CGGTCCGAGT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LAlaArgMet ProAspGly GluAspLeuVal ValThrHis GlyAspAla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14701 AGGCGCGCAT GCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC TCCGCGCGTA CGGGCTGCCG CTCCTAGAGC AGCACTGGGT ACCGCTACGG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ CysLeuProAsn IleMetVal GluAsnGly ArgPheSerGly PheIleAsp* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14751 TGCTTGCCGA ATATCATGGT GGAAAATGGC CGCTTTTCTG GATTCATCGA ACGAACGGCT TATAGTACCA CCTTTTACCG GCGAAAAGAC CTAAGTAGCT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *CysGlyArg LeuGlyValAla AspArgTyr GlnAspIle AlaLeuAlaThr* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14801 CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA GACACCGGCC GACCCACACC GCCTGGCGAT AGTCCTGTAT CGCAACCGAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *TArgAspIle AlaGluGlu LeuGlyGlyGlu TrpAlaAsp ArgPheLeu ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14851 CCCGTGATAT TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GGGCACTATA ACGACTTCTC GAACCGCCGC TTACCCGACT GGCGAAGGAG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ValLeuTyrGly IleAlaAla ProAspSer GlnArgIleAla PheTyrArg* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14901 GTGCTTTACG GTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG CACGAAATGC CATAGCGGCG AGGGCTAAGC GTCGCGTAGC GGAAGATAGC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LeuLeuAsp GluPhePhe*** Stop ~~~~~~~~~~~~~~~~~~~~ 14951 CCTTCTTGAC GAGTTCTTCT GAGCGGGACT CTGGGGTTCG AAATGACCGA GGAAGAACTG CTCAAGAAGA CTCGCCCTGA GACCCCAAGC TTTACTGGCT 15001 CCAAGCGACG CCCAACCTGC CATCACGAGA TTTCGATTCC ACCGCCGCCT GGTTCGCTGC GGGTTGGACG GTAGTGCTCT AAAGCTAAGG TGGCGGCGGA 15051 TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC CGGCTGGATG AGATACTTTC CAACCCGAAG CCTTAGCAAA AGGCCCTGCG GCCGACCTAC 15101 ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCC ACCCCGGGAG TAGGAGGTCG CGCCCCTAGA GTACGACCTC AAGAAGCGGG TGGGGCCCTC 15151 ATGGGGGAGG CTAACTGAAA CACGGAAGGA GACAATACCG GAAGGAACCC TACCCCCTCC GATTGACTTT GTGCCTTCCT CTGTTATGGC CTTCCTTGGG 15201 GCGCTATGAA CGGCAATAAA AAGACAGAAT AAAACGCACG GTGTTGGGTC CGCGATACTT GCCGTTATTT TTCTGTCTTA TTTTGCGTGC CACAACCCAG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15251 GTTTGTTCAT AAACGCGGGG TTCGGTCCCA GGGCTGGCAC TCTGTCGATA CAAACAAGTA TTTGCGCCCC AAGCCAGGGT CCCGACCGTG AGACAGCTAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15301 CCCCACCGAG ACCCCATTGG GGCCAATACG CCCGCGTTTC TTCCTTTTCC GGGGTGGCTC TGGGGTAACC CCGGTTATGC GGGCGCAAAG AAGGAAAAGG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15351 CCACCCCACC CCCCAAGTTC GGGTGAAGGC CCAGGGCTCG CAGCCAACGT GGTGGGGTGG GGGGTTCAAG CCCACTTCCG GGTCCCGAGC GTCGGTTGCA ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15401 CGGGGCGGCA AGCCCTGCCA TAGCCACGGG CCCCGTGGGT TAGGGACGGC GCCCCGCCGT TCGGGACGGT ATCGGTGCCC GGGGCACCCA ATCCCTGCCG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15451  GGATCGCGGC CC        CCTAGCGCCG GG       ~~~~~~~~~~~~~ 

1. A method of producing a protective immune response in a human subject comprising administering to the subject an effective amount of lung cancer cells transfected with a eukaryotic expression vector derived from the bovine papilloma virus comprising a nucleic acid encoding CD80 (B7.1) and with a eukaryotic expression vector derived from the bovine papilloma virus comprising a nucleic acid encoding an HLA antigen.
 2. The method of claim 1, wherein the eukaryotic expression vector comprises a nucleic acid encoding a mouse metallothionein-I promoter (MTI).
 3. The method of claim 1, wherein the eukaryotic expression vector comprises a nucleic acid encoding a G418 resistance gene or histidinol resistance gene.
 4. The method of claim 1, wherein the HLA antigen is selected from HLA A1, HLA A2, HLA A3 and HLA A27.
 5. The method of claim 1, wherein the lung cancer cell is an adenocarcinoma.
 6. The method of claim 2, wherein the HLA antigen is selected from HLA A1, HLA A2, HLA A3 and HLA A27.
 7. The method of claim 2, wherein the lung cancer cell is an adenocarcinoma.
 8. The method of claim 1, wherein the lung cancer cells are administered more than once.
 9. A method of producing a protective immune response in a human subject comprising administering to the subject an effective amount of lung cancer cells transfected with a eukaryotic expression vector derived from the bovine papilloma virus comprising a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen.
 10. The method of claim 9, wherein the eukaryotic expression vector comprises a nucleic acid encoding a mouse metallothionein-I promoter (MTI) and a nucleic acid encoding a cytomegalovirus (CMV) promoter.
 11. The method of claim 9, wherein the eukaryotic expression vector comprises a nucleic acid encoding a G418 resistance gene or histidinol resistance gene.
 12. The method of claim 9, wherein the HLA antigen is selected from HLA A1, HLA A2, HLA A3 and HLA A27.
 13. The method of claim 9, wherein the lung cancer cell is an adenocarcinoma.
 14. The method of claim 10, wherein the HLA antigen is selected from HLA A1, HLA A2, HLA A3 and HLA A27.
 15. The method of claim 10, wherein the lung cancer cell is an adenocarcinoma.
 16. The method of claim 9, wherein the lung cancer cells are administered more than once.
 17. A population of lung cancer cells genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen, wherein at least at least 70% of the cells co-express CD80 (B7.1) and an HLA antigen.
 18. The population of lung cancer cells of claim 17, wherein the HLA antigen is selected from HLA A1, HLA A2, HLA A3 and HLA A27.
 19. The population of lung cancer cells of claim 17, wherein the lung cancer cell is an adenocarcinoma.
 20. A method of manufacturing a vaccine against lung cancer comprising genetically modifying a population of lung cancer cells to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen, wherein at least at least 70% of the cells co-express CD80 (B7.1) and an HLA antigen.
 21. The method of claim 20, wherein the HLA antigen is selected from HLA A1, HLA A2, HLA A3 and HLA A27.
 22. The method of claim 20, wherein the lung cancer cell is an adenocarcinoma. 